Datasets:

Galahad3x

/

QADatasetForPatho

like 0

100

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

Which contaminants or viruses or bacteria were found?

101

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

Which were the symptoms?

102

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What did the patients have?

103

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What were the first steps?

104

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What did they do to control the problem?

105

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What did the local authorities advise?

106

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What were the control measures?

107

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What type of samples were examined?

108

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What did they test for in the samples?

109

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What is the concentration of the pathogens?

110

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What steps were taken to restore the problem?

111

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What was done to fix the problem?

112

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What could have been done to prevent the event?

113

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

How to prevent this?

114

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What were the investigation steps?

115

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What did the investigation find?

116

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

How was the infrastructure affected?

117

A community-wide acute diarrheal disease outbreak associated with drinking contaminated water from shallow bore-wells in a tribal village

Abstract Background: In 2016, India reported 709 acute diarrheal disease (ADD) outbreaks (> 25% of all outbreaks). Tribal populations are at higher risk with 27% not having accessibility to safe drinking water and 75% households not having toilets. On June 26, 2017 Pedda-Gujjul-Thanda, a tribal village reported an acute diarrheal disease (ADD) outbreak. We investigated to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods: We defined a case as ≥3 loose stools within 24 h in Pedda-Gujjul-Thanda residents from June 24–30, 2017. We identified cases by reviewing hospital records and house-to-house survey. We conducted a retrospective cohort study and collected stool samples for culture. We assessed drinking water supply and sanitation practices and tested water samples for faecal-contamination. Results: We identified 191 cases (65% females) with median age 36 years (range 4–80 years) and no deaths. The attack-rate (AR) was 37% (191/512). Downhill colonies (located on slope of hilly terrains of the village) reported higher ARs (56%[136/243], p < 0.001) than others (20%[55/269]). Symptoms included diarrhea (100%), fever (17%), vomiting (16%) and abdominal pain (13%). Drinking water from five shallow bore-wells located in downhill colonies was significantly associated with illness (RR = 4.6, 95%CI = 3.4–6.1 and population attributable fraction 61%). In multivariate analysis, drinking water from the shallow bore-wells located in downhill colonies (aOR = 7.9, [95% CI =4.7– 13.2]), illiteracy (aOR =6, [95% CI = 3.6–10.1]), good hand-washing practice (aOR = 0.4, [95%CI = 0.2–0.7]) and household water treatment (aOR = 0.3, [95%CI = 0.2–0.5]) were significantly associated with illness. Two stool cultures were negative for Vibrio cholerae. Heavy rainfall was reported from June 22–24. Five of six water samples collected from shallow bore-wells located in downhill colonies were positive for faecal contamination. Conclusion: An ADD outbreak with high attack rate in a remote tribal village was associated with drinking water from shallow downhill bore-wells, likely contaminated via runoff from open defecation areas after heavy rains. Based on our recommendations, immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families, and as long-term public health measures construction of house-hold latrines and piped-water supply initiated. Keywords: Acute diarrheal disease, Outbreak, Bore-well, Tribal Introduction Globally there are an estimated 1.7 billion cases and 2.2 million deaths from acute diarrheal disease (ADD) every year [1]. In India, the burden is particularly high with more than 13.9 million cases reported in 2016 and 709 ADD outbreaks reported accounting to more than 25% of all outbreaks [2, 3]. Lack of access to safe drinking water and basic sanitation are the leading causes of ADD burden globally and in India. It is estimated that globally 58% of ADD deaths are attributed to inadequate drinking water, sanitation and hygiene [4]. The WHO/UNICEF Joint Monitoring Program for Water Supply, Sanitation and Hygiene (JMP) 2017 report revealed that 844 million people worldwide lack access to basic drinking-water service and 2.3 billion lack basic sanitation services, while 892 million still practiced open defecation [5]. The National Family Health Survey (NFHS-4, 2015–16) reported that in India only 52% of urban households and 18% of rural households have piped water supply, and the main source of water supply among rural households is bore-wells or tube-wells (51%). It has been estimated that 39% of households in India (54% among rural households) have no toilet facility and practicing open defecation [6]. The “indigenous†populations are socially, culturally and economically isolated and usually lack access to basic drinking-water and sanitation services. Therefore, they are vulnerable to ADD outbreaks and other emerging and re-emerging diseases [7]. The United Nations estimates that there are 370 million indigenous people existing across 90 countries of the world. They constitute 5% of the world population but 15% of the poorest [8]. India alone houses more than 705 such indigenous groups termed as Scheduled Tribes. As per the Census 2011, the total Scheduled Tribe population of India is 10.43 crore with a significant proportion of them living in rural areas [9]. On June 26, 2017, Kama-reddy district of Telangana state reported 55 ADD cases from the Pedda-GujjulThanda village. We conducted the outbreak investigation to describe the epidemiology, identify risk factors, and provide evidence-based recommendations. Methods Setting Pedda-Gujjul-Thanda village is a small tribal village with a total population of 563. The village is remotely located as an isolated community with a hilly terrain and is resource-limited with poor accessibility to sanitation and hygiene facilities. The nearest health care facility available for the residents is located at a distance of 10 km from the village. Case definition We defined a case as three or more loose stools within 24 h in a resident of the Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Case finding To find cases, we reviewed medical records of local health care facilities accessed by village residents in the nearby town. We conducted a medical camp in the village during the outbreak period for five days. We conducted a house-to-house survey in the village to find more cases, which are niether reported to health facility nor medical camp. Retrospective cohort study We conducted a retrospective cohort study to identify risk factors associated with illness. We defined the cohort as residents of Pedda-Gujjul-Thanda village from June 22, 2017 to July 2, 2018. Village resident was the unit of analysis. For data collection, we trained five teams of local paramedical staff. Using a pre-structured questionnaire, we collected data on demographic characteristics and risk factors related to drinking water, sanitation and hygiene. Good hand-washing practice was defined as reported washing of hands with soap and water every time after defecation and before eating. A bore-well less than 30-m-deep, as assessed from the records of village administration, was considered a shallow bore-well. Laboratory and environmental investigations Two stool samples were collected by the treating physician from admitted patients on the first day of hospital admission and transported to the state reference laboratory within two hours in Cary-Blair transport medium. The samples were cultured for Vibrio cholerae, Salmonella and Shigella on nutrient agar, MacConkey agar and deoxycholate citrate agar. Enteric pathogens were identified by biochemical reaction and by agglutination with anti-sera. We collected details of recent rainfall and conducted an environmental survey with household as sampling unit to assess drinking water, sanitation and hygiene practices. We assessed availability of residual chlorine in all village bore-wells and tested four of five bore-wells in the most affected colonies for faecal contamination by H2S method in field. Water was filled up to the ‘fill line’ of the sample bottle and incubated at room temperature (250–370 C) for 36–48 h and observed for colour change in the medium. A water sample was suspected to be contaminated with faecal matter, if it turned black [10, 11]. Because of limited supplies, we were unable to assess the fifth bore-well. Data analysis We analysed the data to describe the occurrence of cases over time, place, and person. We calculated relative risks (RR) with 95% confidence intervals (CI), population attributable risk percentages and conducted multiple logistic regression analysis with the dependent variables including consumption of shallow-downhill bore-well water, report of visible contaminants like mud in drinking water, illiteracy, household water treatment and good hand-washing practice. We used Epi Info version 7.2 for statistical analysis. Results Descriptive epidemiology We identified 191 ADD cases (65% females), with a village attack rate (AR) of 37% (191/512). The attack rate increased with age, with highest among > 60-year age group (55%) and lowest among children under-10 years (11%) (Table 1). No deaths were reported. In addition to diarrhea, cases presented with fever (17%), vomiting (16%) and abdominal pain (13%). 72% (138/191) cases reported to health care facilities and the medical camp conducted in the village. Among the 191 cases, 159 (83%) had mild illness treated with oral rehydration solution; 30 (16%) had moderate dehydration treated with intravenous fluids on out-patient basis, and 2 (1%) with severe dehydration were admitted in the district hospital for treatment with antibiotics (metronidazole and ciprofloxacin) and intravenous fluids. Cases started reported on June 26, 2017, with onset of symptoms from 24 June 2017. Maximum cases were reported on June 27, 2017, and no new cases were reported after June 30, 2017 (Fig. 1). The tribal population in the village had four sub-tribes namely Katroth, Badhawath, Nenawath, Baromath who resided in seven geographically demarcated colonies (labelled as A to G). Katroth sub-tribe resided in colonies A, B and G; Badhawath in colonies C and D; Nenawath in colony E and Baromath in colony F (Table 2). Colonies B and C had higher attack rates (65 and 47% respectively) as compared to other colonies (Fig. 2). Retrospective cohort study Among 563 village residents, 512 (91%) participated in the study. Among the 512 participants, median age was 28 years (range 1–80 years) with 52% females; 50% reported as illiterate with agriculture as the main source of livelihood for 76%. We analysed possible risk factors associated with ADD (Table 3). Drinking water from bore-well groundwater (vs canned water) was found significantly associated with ADD (RR = 12.7; 95% CI = 1.8–87.4). However, only 32 (6%) residents in the village used canned water and bore-well groundwater was the predominant source of water supply. Therefore, we analysed the water sources further, by location and type of bore-wells. Residents who used any of the five shallow bore-wells located downhill were significantly at higher risk (RR = 4.6; 95% CI = 3.4–6.1) and deep bore-wells were protective (RR = 0.4; 95% CI = 0.2–0.9). Report of visible contaminants like mud in drinking water (aOR = 4; 95% CI = 2.1–7.6) and illiteracy (aOR = 3.6; 95% CI = 3.5–10.1) were significantly associated with illness; and household water treatment (done either by boiling or use of candle filters) (aOR = 0.4; 95% CI = 0.2–0.7) and good hand-washing practice (aOR = 0.2; 95% CI = 0.1–0.5) were found protective. Laboratory and environmental results Stool samples collected from two hospitalized cases showed no growth for Vibrio cholerae, Salmonella and Shigella on culture. Among 110 households, 100 (91%) were available for environmental survey. Among the 100 houses surveyed, 79 (79%) were kutcha (low quality) type, made of mud, thatch and other low-quality material. Only 5 (5%) households had a designated toilet at home while the remaining 95 (95%) practiced open defecation at a site located on the slope of the hill behind the downhill colonies B and C (Figs. 2 and 3). Bore-wells were the main source of drinking water supply for 93 (93%) households. There were two deep borewells provided by the village administration and 17 shallow type bore-wells privately constructed by village residents. Five of these 17 (30%) shallow bore-wells were located in colonies B and C, on the downhill slope below the open defecation site. Plastic pipelines from the shallow wells were improperly installed with leakages at multiple points. There was no facility at source, for chlorination or any other mode of purification. Thirty households (30%) treated the water before consumption either by boiling or by use of candle filters. There was no routine drinking water surveillance in place by any authority for assessing the quality and fitness for drinking water. There was no residual chlorine found in any water samples. Three of four drinking water samples from bore-wells of most affected colonies (B and C) indicated faecal contamination by H2S field testing. There was heavy rainfall (average 65 mm in a day) from 22 to 24 June 2017. Prevention and control measures undertaken to contain the outbreak The village residents were discouraged from using shallow bore-well water and were provided with safe canned drinking water until all leakages were secured. Leakages in water supply from the bore-wells were identified and secured. Chlorine tablets were distributed for household level water disinfection. We informed the residents to avoid open defecation near drinking water sources and residential premises. Public health staff conducted health education daily to improve awareness among the villagers about water, sanitation, and hygiene. After active implementation of these control measures, cases declined rapidly in the village (Fig. 1). Discussion A rapid systematic epidemiological investigation of this outbreak identified water contamination points and likely mode of contamination. Based on these findings and our recommendations, the local health department instituted immediate public health actions including repair of leakages at contaminated water sources and alternative supply of purified canned drinking water to families. Effective implementation of public health measures limited the exposure of the community to contaminated water source resulting in rapid containment of the outbreak. Waterborne disease outbreaks tend to have cases spread over a time-period due to ongoing exposure to the contaminated water. In contrast, the pattern of epidemic curve in the present waterborne disease outbreak resembled that of food-borne with a point source exposure. Heavy rains contributed to the run-off of water from the open defecation site into the ground water of shallow wells located on slopes of hilly terrain resulting in heavy contamination and sudden rise of cases. Rapid control measures in the small village, implemented effectively within a short period of time, may have led to rapid decline of cases. The available epidemiological evidence also did not support generation of hypothesis of food-borne origin of the outbreak. In an outbreak reported among school children in Northern Greece in 2012, investigation revealed a waterborne viral gastroenteritis outbreak with a point source pattern, due to consumption of heavily contaminated water from a tap, which was not in use for two weeks during Christmas vacation [12]. Attack rate was high in this outbreak (37%), possibly due to exposure to high pathogen load subsequent to gross faecal contamination of water sources. In the absence of other alternative water sources, this tribal community was exclusively dependent on the contaminated water source for drinking, therefore exposing a large section of the community to risk. Geetha et al. analysed 32 diarrheal outbreaks in south India in non- tribal communities and reported lower attack rates varying from 0.6 to 21.5% [13]. However, tribal populations in India such as in Pedda-Gujjul-Thanda are marginalized with poor availability of WASH facilities [14]. This vulnerable tribal population continues to be at higher risk for ADD outbreaks with 27% not having access to safe drinking water and 75% of households not having toilets [15]. They need special assistance schemes from the government to enable them overcome poor accessibility to WASH facilities and secure healthy living [16]. Due to inadequate availability of communally managed safe public water points by the local authority, this community in Pedda-Gujjul-Thanda village was dependent on privately constructed shallow bore-wells for water supply. These are economical but likely to be unsafe. In this outbreak, open defecation site was present on the downhill slope in proximity to the residential premises and water resources, increasing the risk of drinking water contamination. Among the entire village population, 61% of ADD cases were attributable to drinking water from the ‘shallow downhill bore-wells’ (Population Attributable Fraction 61%), which was also evident from rapid outbreak containment following the elimination of exposure to this single risk factor. Since this exposure factor is amenable to long-term public health intervention, permanent elimination of shallow downhill borewells as water source was recommended, replacing them with properly secured deep bore-wells. Shallow bore-wells are known for their susceptibility to contamination from surface land-use activities [17, 18]. Studies have found levels of E. coli and enteric viruses to be high in shallow sources of ground water especially when they are in close proximity to polluting sources [19–21]. Consumption of ground water from shallow bore-wells with no purification facility increases the risk of diarrhea outbreaks manifold [22, 23]. A metaanalytic study of water-borne diarrheal disease outbreaks in China reported that 78 of 85 (92%) outbreaks (between year 1987 to 2014) were due to poor sanitary conditions of wells with lavatories/septic tanks nearby and lack of purification facilities [24]. In developed countries and urban areas of developing countries, as water supply and sanitation have improved dramatically over a period of time, such outbreaks were rarely reported in the recent past. The largest E. coli O157 outbreak in United States occurred in 1999 at a county fair (781 ill persons and 2 deaths) was due to groundwater source from a temporary unregulated well at the fairground [25]. Our findings have implication for India’s progress towards United Nation’s Sustainable Development Goal (SDG) 6 and India’s nation-wide campaign ‘Swachh Bharat Mission (SBM)’ to ensure availability and management of water and sanitation for all. SDG 6 aims at achieving universal access to basic sanitation service by 2030; and it has been reported that between 2000 and 2017, the proportion lacking even a basic sanitation service decreased from 44 to 27% [26]. SBM aims to achieve an open-defecation free status in rural areas through the construction of household-owned and community-owned toilets and establishing an accountable mechanism of monitoring toilet use. In 2015 in India, around 524 million (39%) practiced open defecation. However, under the SBM mission, due to increase in ‘households with toilets’ only 19 million (1.4%) practiced open defecation in January 2019 [5, 27]. There has also been a 71.58% increase in ‘households with toilets’ from October 2014 to October 2019 in rural areas of the Telangana state in India [27]. The tribal community initially obstructed the effective delivery of health care services; however, after involvement of the local stakeholders and tribal leaders, the acceptance towards medical treatment and community health services improved. Notwithstanding, most of the patients were still reluctant and did not consent for giving stool specimens for laboratory diagnosis. Establishing a rapport with the reticent tribal community was a major challenge faced by the outbreak investigation team. Lack of microbiological aetiology confirmation of the outbreak remained a limitation of the investigation due to limited stool samples and laboratory-capacity constraints of the remote area. Recognizing the pivotal importance of SDGs, national health policy of India (2017) has set the health-related cross-sectorial goal “access to safe water and sanitation to all by 2020†[28]. Greater political and financial commitment towards resource-limited remote tribal areas with effective community mobilization is required to accelerate the public health interventions to improve WASH and to prevent ADD outbreaks in the future. Conclusion This was a community-wide acute diarrheal disease outbreak with high village attack rate in a remote tribal village of Telangana with poor availability of safe water, sanitation and hygiene (WASH) facilities. A rapid and systematic epidemiological investigation identified drinking of faecal-contaminated water from the shallow borewells as the leading cause for this outbreak. These borewells were likely contaminated from runoff after rain from open defecation areas located on a downhill slope. Prompt and targeted public health action contained the number of cases.

What were the infrastructure complaints?

118

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What happened?

119

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What was the event?

120

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

When did this happen?

121

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

When did this event start?

122

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What is the date of this event?

123

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

How long was the event?

124

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

How long did the event last?

125

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

In which street did this happen?

126

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

In which city did this happen?

127

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

In which region did this happen?

128

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

In which country did this happen?

129

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

Where did this happen?

130

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What caused the event?

131

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What caused the contamination of the tap water with river water?

132

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What was the cause of the event?

133

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What source started the event?

134

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

How was the event first detected?

135

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

How many people were ill?

136

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

How many people were hospitalized?

137

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

How many people were dead?

138

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

Which contaminants or viruses or bacteria were found?

139

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

Which were the symptoms?

140

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What did the patients have?

141

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What were the first steps?

142

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What did they do to control the problem?

143

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What did the local authorities advise?

144

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What were the control measures?

145

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What type of samples were examined?

146

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What did they test for in the samples?

147

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What is the concentration of the pathogens?

148

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What steps were taken to restore the problem?

149

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What was done to fix the problem?

150

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What could have been done to prevent the event?

151

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

How to prevent this?

152

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What were the investigation steps?

153

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What did the investigation find?

154

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

How was the infrastructure affected?

155

A Diarrhoeagenic Enteropathogenic Escherichia coli (EPEC) Infection Outbreak That Occurred among Elementary School Children

Abstract Background: In response to the notification made by an elementary school authority that reported a number of elementary school children being absent in three schools as a result of gastroenteritis symptoms on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea, an epidemic investigation was carried out to determine the extent, cause, and source of the outbreak in order to prevent secondary cases and make recommendations to prevent future recurrences. Methods: In this epidemiologic study, a total of 106 human subjects (school children, staff members, and cooks) who had consumed the possibly contaminated foodstuffs were enrolled retrospectively. Human specimens from clinically defined cases, food and drinks, supply and storage of them, and environmental and sanitary conditions were also assessed by observation, laboratory tests, and survey questionnaires—where and whatever applicable. The attack rate and positive rate for human specimens were first presented followed by the calculation of the relative risk ratio (RR) with 95% CI (confidence intervals) in order to identify the exposure and outcome relationships. Results The attack rate was 12.26% (13/106) for those who had ingested the food items at the three schools and the positive rate of enteropathogenic Escherichia coli (EPEC) was 15.38% (2/13). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang and seasoned cucumber and chives were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. In addition, within the human specimens as well as the water and environmental samples different strains of diarrhoeagenic enteropathogenic Escherichia coli (EPEC) were detected. Conclusions Provision of safe and wholesome water access to all elementary schools by concerned authorities, especially during the likely seasons of water source contamination, as well as health education promotion about foodborne outbreaks to all school stakeholders is therefore recommended. Keywords: diarrhoea; enteropathogenic Escherichia coli outbreak; epidemiology; school feeding 1. Introduction Food and waterborne illnesses are worldwide-prevalent public health problems [1,2]. Food poisoning is an acute gastroenteritis caused from ingestion of food or drinks with either microbial or non-microbial contamination. Acute gastroenteritis is one of the leading causes of morbidity and mortality [2]. The World Health Organization (WHO) estimated that every year 600 million (almost 1 in 10 people) fall in sick and nearly 420,000 deaths occurs worldwide as a result of contaminated food consumption, giving rise the loss of 33 million healthy lives (DALYs) [3]. Out of this number, children under 5 years of age hold 40% of the morbidity and 125,000 mortalities every year. A report shows that more than 200 diseases ranging from diarrhoea to cancer are caused due to food poisoning that can lead to losses in national economy, increased health care cost, and impede the development of trade and tourism [3]. Likewise, contaminated water consumption is one of the major causes of waterborne human infections [4]. Waterborne diarrhoeal disease claims 2 million deaths worldwide each year, mostly in children below 5 years of age. Of the total world population, only approximately 663 million people are reported to have consistent access to improved drinking water sources [4,5]. The major features of food poisoning are headache, intense thirst, acute vomiting, giddiness, diarrhoea, slow pulse, cramps and rigors, colicky pain, cold, and clammy skin [2]. Salmonella, Campylobacter, Vibrio cholerae, and enterohaemorrhagic Escherichia coli are the most common foodborne pathogens. The usual sources of these pathogen are raw or undercooked foodstuffs, such as egg, meat, milk or milk products, fruits and vegetables, and water [3]. Equally, several waterborne gastroenteritis epidemics have been found to be generated by diarrhoeagenic Escherichia coli (DEC), Gram-negative bacteria, which are divided into five groups: enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) [6,7]. Food poisoning and waterborne outbreaks in non-community places, such as schools, colleges, restaurants, and hotels, are found to be linked with several microbial and non-microbial factors, likely due to the contamination of food and drinks. Low pressure conditions of water, low residual chlorine concentration, and broken-down piped water supply are responsible for the intrusion of pathogenic microorganisms in water, while food-related factors, such as food handling policy, undercooked, improper labelling, and cleaning of raw foodstuffs, can provide an opportunity for microbes to invade into the food items [3,8,9]. Among many other sources, the chief sources of water, food, and drinks contamination usually reported are human sewage, animal wastes, overly used toxic agricultural chemicals, and pesticides that are mixed up into the water supply system [3,4,10]. Removal of biological and chemical contamination from drinking water prior to consumption is a crucial step in water safety [11]. Managing the quality of water from all sources, including public water and groundwater, by a multistep treatment system can reduce the risk of infection in the human body [12]. Similarly, standard food safety policy, appropriate handling, proper cooking, cleaning, training to food handlers, as well as good coordination between governments, producers, and consumers help ensure food safety [3]. Although there are infrequent reports from developed countries about the outbreak of food and waterborne infections associated with EHEC infections as the etiological agent, multiple bacterial and viral strains may have been involved for such outbreaks [3,7]. Despite having less attention to EHEC infections occurring as a result of consumption of contaminated food and water in developed countries, especially in non-community settings, such findings are important for the evidence-based strategy formulation in order to reduce and prevent recurrence of diarrhoeal illnesses. This study aimed to identify the extent, cause, and source of an outbreak—through epidemic investigation—that occurred among elementary school children in Gyeongsangbuk-Do Province, South Korea, to prevent secondary cases and to make recommendations as to the prevention of future recurrences. 2. Materials and Methods 2.1. Study Background and Settings The epidemiological investigation was carried out with the receipt of the notification from a school authority that a number of elementary school children were absent in school as a result of symptoms of gastroenteritis diarrhoea, fever, and abdominal pain on 4 July 2018, in Gyeongsangbuk-Do Province, South Korea. Administratively, South Korea is comprised of 17 first-tier administrative divisions: 6 metropolitan cities, 1 special city, 1 special autonomous city, and 9 provinces, including one special autonomous province. These are further subdivided into a variety of smaller entities, including cities (Si), counties (Gun), districts (Gu), towns (Eup), townships (Myeon), neighbourhoods (Dong), and villages (Ri) [13]. Of the 106 human subjects who consumed the possibly contaminated foodstuffs in three elementary schools (one main elementary and another two branches) from the city (Si) of Gyeongsangbuk-Do Province of South Korea, 13 developed food poisoning (elementary school children and teachers). The study team consisted of epidemiologists, laboratory personnel to collect sample specimens from the affected subjects, and a food hygienist to check and collect the suspected food and drinks causing the food poisoning, as well as environmental samples. 2.2. Epidemiological Investigation A case of food poisoning was defined as those who were having 3 episodes of diarrhoea within 24 h or 2 diarrhoeal episodes, and one or more symptoms of nausea and vomiting, abdominal pain and fever, as per the guideline suggested by Korea Centre for Disease Control and Prevention (KCDC) [14]. Similarly, the cases of food poisoning were confirmed with cultures positive for EPEC. A total of 13 cases occurred during the period of 26 June to 3 July 2018 and were analysed retrospectively to identify the risk factors associated with food and drinks and environmental stuffs. All relevant study subjects (school children and their parents, school teachers, cook workers, and food distributors) were interviewed using in-built epidemiological case-sheets as suggested by KCDC to collect a wide range of information about the biosocial aspects as well as the food and drinks consumed, processed, prepared, and delivered, and exposure to other environmental and ecological conditions. All the cases developed during the study period were monitored and supervised continuously and follow-up surveillances were conducted twice during the incubation period until the study settings were declared free of disease. In the containment of the outbreak and its prevention to further spread, a number of general and specific measures were applied, such as imparting health education about water and foodborne infections and methods of prevention and control like the use of boiling water and proper hand washing to all of the target population, including users of tap water in neighbouring villages. In addition, to prevent further occurrences, disinfection was carried out in school lunch facilities and restrooms, and the water and sewage establishments were suggested to liaise with the school and the village’s waterworks management. 2.3. Environmental and Ecological Assessment A study team examined all the possible environmental and ecological factors to identify the causes of outbreak. In this course of action, the food hygiene officer examined the ingested and stored food items at the school meal centre, and checked for drinking and cooking water, tap water and groundwater in the food service centre, and the chlorine concentration. Moreover, the interior of the kitchen and the inspection status of the cooking chamber were also observed. Food, water, environmental, and other ecological samples were collected and tested to detect the microbes causing the outbreak. 2.4. Microbiological and Biochemical Examination of Human and Environmental Samples The Gyeongsangbuk-do Health Environment Laboratory technologists collected clinical sample specimen from all cases affected. Rectal swabs and stool samples were collected. Thus, collected samples were cultured and tested at the local health authority. Further, these isolates were transferred to the KCDC in order to confirm the causative agents. The food poisoning-causing pathogens tested for were bacteria (16 species): Cholera, Salmonella typhi, Salmonella paratyphi, Shigellosis, enterohemorrhagic E. coli, Salmonella spp., Vibrio parahaemolyticus, enterotoxic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Yersinia enterocolitica, Listeria monocytogenes; and viruses (6 species): Hepatitis A, Rotavirus, Astrovirus, Adenovirus, Norovirus (G1, G2), and Sapovirus. In case of food and drinks and environmental specimens, the following possible pathogens and biochemical investigation were performed: Drinking water (9 kinds): general bacteria, total coliform group, faecal coliform group, ammonia–nitrogen (NH3-N), nitrate–nitrogen (NO3-N), free residual chlorine, potassium permanganate consumption, chlorine ion, and sulfuric acid ion. 2.5. Statistical Analyses The Chi-square test to investigate the different food and drinks and environmental risk factors was employed. Thus, between exposure and non-exposure cohorts, the relative risk (RR) with 95% confidence interval (CI) for all samples examined were calculated. A p-value < 0.05 was set as statistically significant. All statistical analyses were conducted using SPSS for Windows (version 22.0, SPSS Inc. Chicago, IL, USA). 2.6. Ethics This study falls within the rules and regulations of the Korean Bioethics and Safety Act (Article, 15) and Infectious Disease Control and Prevention Act (Article 18) set by the Korea Center for Disease Control and Prevention, Ministry of Health and Welfare, and the Provincial Government of South Korea; therefore, an additional ethical approval from the institutional review board or ethical committee was not necessary. All personal details were removed prior to data analysis. 3. Results 3.1. Descriptive Results The incidence rate according to case definition was 12.26% (13/106) of those who had ingested the food items at three elementary schools and the positive rate of EPEC was 15.38% (2/13). Among the clinically defined cases, 9/13 (69.23%) were male, while the laboratory-confirmed two cases were one male and another female. Of total 13 defined cases, the percent reported to have diarrhoea followed by fever was 76.9%, abdominal pain 53.8%, nausea in 15.4%, and the lowest percentage was chills (7.7%). Figure 1 shows that the trend of the cases among the study subjects in the study area. Of the 13 cases, nearly half (46.1%) of the cases occurred after the third day of exposure (within the one incubation period), resembling a point-source epidemic. 3.2. Results of Laboratory Tests of Human Specimens Table 1 shows the laboratory test results of the human specimens. Of the 13 clinically confirmed cases of food poisoning, 15.38% (2/13) were confirmed to have EPEC O139, ONT in the human specimens; both of them had ingested the possibly contaminated foodstuffs. 3.3. Diarrhoeagenic EPEC Infection Outbreak Associated with Consumption of Water-Contaminated Food Items Of number of food items consumed that were obtained through personal interviews of the affected subjects, the cucumber tofu that was supplied in the meals was found to be associated with the food poisoning (Table 2). The relative risk (RR) of developing food poisoning of those who consumed the cucumber chili with ssamjang on 27 June and seasoned cucumber and chives on June 28 were 4.55 (95% CI 1.05–19.54) and 9.20 (95% CI 1.24–68.22), respectively. 3.4. Results of Laboratory Tests of Environmental Specimens Table 3 demonstrates the 16 types of food poisoning-causing bacteria and nine types of water-borne bacteria tested for in 39 food items, as well as other environmental and ecological specimens. It was revealed that the cooking water (from the village water supply) and dishwashing water (from the village water supply) were found to have EPEC O87. Regarding the concentration of the coliforms, both the cooking and drinking water (from the village water supply) had both total coliforms (+) and faecal coliforms (+), while the main school water purifier (from the village water supply) had only total coliforms (+). 4. Discussion This outbreak investigation sought epidemiologic, laboratory and environmental evidences in the occurrence of the outbreak of diarrhoeagenic EPEC infection in three elementary school’s children in the Gyeongsangbuk-Do province of South Korea. We observed the clinically defined cases [14]. (those having at least three episodes of diarrhoea within 24 h or two and more diarrhoeas and one or more symptoms of nausea, diarrhoea, and fever as defined by the KCDC) of those who had consumed foods served at schools from 25 June to 29 June 2018. The attack rate among clinically confirmed cases was 12.26% and the laboratory confirmed cases with EPEC was 15.38%, while nearly half (46.1%) of those of clinically confirmed cases occurred on the third day of exposure (within the one incubation period), indicating that the outbreak is a point-source epidemic. Previous similar water and foodborne outbreaks caused by EPEC occurred in similar school settings of South Korea [15,16], which reported more than twice higher attack rates compared with this study; all these studies, including the current one, suggest that EPEC outbreaks in South Korean school settings should not be underestimated. In fact, Intestinal pathogens, such as enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) are less common in developed countries, and are usually endemic in developing countries [17], and therefore, less attention is given in developed countries to such pathogens unless the diarrhoeagenic outbreak is more obviously prevalent and reported. Accordingly, in South Korea, diarrheal diseases caused by EPEC has not been listed as a national notifiable disease. However, the reason why it is important in terms of public health significance in South Korea, is that it is difficult to recognize unless there is massive prevalence of diarrhoeal diseases, and that of the symptoms of EPEC infections are reported to be weaker and illnesses are sporadic compared to other diarrhoeal illnesses [18]. In addition, since gastrointestinal illnesses occurring as a result of faulty water supply systems is on the decline in developed countries, it has been of less concern and thus overlooked in conditions favouring water contamination, especially in rural settings during rainy seasons [19]. In our study, the human cases specimen investigation confirmed EPEC O139, ONT strains in those clinically confirmed cases who were exposed to foods served at school. Of the 29 foodstuffs consumed during the period of 25 June to 29 June 2018, cucumber chili with ssamjang and seasoned cucumber and chives posed a higher relative risk (RR) of giving food poisoning. The RR for developing food poisoning of those who consumed the cucumber chili with ssamjang on June 27 and Seasoned cucumber and chives on 28 June were 4.55 and 9.20, respectively, compared to their counterparts of non-exposure. Further, our environmental and ecological investigation revealed that cooking water and dishwashing water (from village water supply system) used in elementary schools were found to have EPEC O87. In South Korea, we generally believe that the centrally controlled water supply system is well established like in other developed countries, but in rural areas where there is no centrally controlled water supply network, and the stability of the simplified wide-area water supply needs to be managed. In recent years, even in the case of a simplified wide-area water supply, an automatic chlorine injector is installed and well maintained, but it needs be checked and managed periodically, and would need to be especially well managed when there is heavy rainfall over a long period, as well as during a draught that can lead to the reduction in efficacy of the chlorine used in the water supply system. In this investigation, during the period of 26~27 June, 2018, there was heavy rainfall all around the current settings, which we assume might have contaminated the source of the drinking water and lessened the chlorine concentration in the piped water supply system. In the current study settings, the foodstuffs were prepared in one main elementary school and it was supplied to the other two branches of the elementary schools. Thus, the same foodstuffs were served during the stated period to all three schools, and the water supply systems were different one another, but cases were observed in all three schools; it could, therefore, be logically assumed that the foodstuffs could have been prepared using contaminated water. Thus, considering the type of food exposure, the incubation period, recommended case definition, human specimen report, and environmental laboratory investigation, we logically concluded that the diarrhoeagenic outbreak that occurred among those three elementary school children was caused by EPEC infection as a result of water-contaminated foodstuffs consumption [20]. Although investigators attempted with their greater strengths to explore supportive evidence of the EPEC food poisoning, the study should have been considered in the light of some specific limitations. First, our retrospective analysis of the data obtained could not rule out the lower positivity test results of the human specimens despite all school personnel having consumed the same foodstuffs. This could be explained in that the affected individuals had already been treated with antibiotics before laboratory investigation started as the school authority reported the event lately because of weekend holidays. Another reason for the higher negative test results in the bacterial culture of the human specimens could be the possibility that the infective period had already been passed out, or the bacteria might not have been cultivated. This is supported by the fact that the intestinal bacteria decrease rapidly as the symptoms improve [18]. In line with the support of this logic, other studies reported positive test result of diarrhoeagenic bacteria among children with diarrhoea ranging from 2.7% to 16%, while it was at 4.0% in children without diarrhoea [21,22]. Secondly, although two of the foodstuffs (the cucumber chili with ssamjang and seasoned cucumber and chives) consumed posed a statistically significant higher RR of causing an outbreak, the same food sample could not be confirmed in culture due to the lack of appropriate food samples (in South Korea, food items can customarily be preserved up to 6 days and are in freeze-dried conditions) because the event was reported late, only on the 4th of July 2018, and outbreak investigation initiated on July 5. In this regard, it should be well advocated to all school stakeholders to notify any of the food- or waterborne illnesses at the soonest possible time to the concerned local health authority, which can help with robust investigation and initiate early interventions. Thirdly, this study might have suffered from recall bias as the data obtained were self-reported, and this may lead to misclassification of exposure. However, the authors attempted to capture the information through the use of already-piloted epidemiological case sheets recommended by the KCDC with different sets of questionnaires for school children, their parents, and the school stakeholders. Moreover, the study could be meaningful to underline how children are the most vulnerable group; a scientific study revealed a strategic point to better understand their health needs and to monitor the quality of care provided to this vulnerable population [23]. 5. Conclusions This epidemic investigation identified that the diarrhoeagenic enteropathogenic Escherichia coli infection outbreak that occurred among elementary school children was due to the consumption of water-contaminated cucumber chili with ssamjang and seasoned cucumber and chives food items. In addition, the human, water, and environmental samples tested detected the strains of diarrhoeagenic enteropathogenic Escherichia coli. South Korean central and provincial governments should ensure safe and wholesome water access to all elementary schools, especially during the likely seasons of water contamination, and the stakeholders of the elementary schools should have been well-advocated with regard to the health education promotion about such water- and foodborne outbreaks, as well as to the importance of early notification of such outbreaks so as to place effective interventions at the incipient stage and prevent future recurrences.

What were the infrastructure complaints?

156

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What happened?

157

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What was the event?

158

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

When did this happen?

159

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

When did this event start?

160

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What is the date of this event?

161

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

How long was the event?

162

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

How long did the event last?

163

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

In which street did this happen?

164

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

In which city did this happen?

165

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

In which region did this happen?

166

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

In which country did this happen?

167

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

Where did this happen?

168

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What caused the event?

169

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What was the cause of the event?

170

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What source started the event?

171

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

How was the event first detected?

172

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

How many people were ill?

173

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

How many people were hospitalized?

174

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

How many people were dead?

175

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

Which contaminants or viruses or bacteria were found?

176

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

Which contaminants or viruses or bacteria were found?

177

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

Which were the symptoms?

178

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What did the patients have?

179

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What were the first steps?

180

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What did they do to control the problem?

181

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What did the local authorities advise?

182

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What were the control measures?

183

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What type of samples were examined?

184

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What did they test for in the samples?

185

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What is the concentration of the pathogens?

186

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What steps were taken to restore the problem?

187

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What was done to fix the problem?

188

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What could have been done to prevent the event?

189

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

How to prevent this?

190

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What were the investigation steps?

191

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What did the investigation find?

192

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

How was the infrastructure affected?

193

A foodborne outbreak of gastroenteritis caused by Norovirus and Bacillus cereus at a university in the Shunyi District of Beijing, China 2018

Abstract Background: On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 suspected students caused of diarrhea and vomiting. The case was investigated, and control measures were taken to prevent further spread. Methods: A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that high risk of food served at the school canteen. We collected information on demographics, refectory records, person to person transmission by uniform epidemiological questionnaire. Risk ratios (RR) and 95% confidence intervals (CI) were calculated. Stool specimens of cases and canteen employees, retained food, water, and environmental swabs were investigated by laboratory analysis. Results: We identified 209 cases (including 28 laboratory-confirmed cases) which occurred from August 29 to September 10. All cases were students, and the average age was 20, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks stall and Rice flour stall on September 1 (RR:3.4, 95%CI 1.5–7.8, and RR:7.6, 95%CI:2.8–20.2), Rice flour stall and Fish meal stall on September 2 (RR:4.0, 95%CI:1.2–13.6, and RR:4.6, 95%CI:1.7–12.5), muslim meal stall on September 4 (RR:2.7, 95%CI:1.3–5.4), Barbeque stall on September 5 (RR 3.0, 95%CI:1.2–7.0) were independently associated with increased risk of disease within the following 2 days. Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive. Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Ten specimens of rectal swabs from canteen employees were positive for norovirus GI, and 2 specimens were positive for Bacillus cereus. Four retained food specimens were positive for Bacillus cereus, and environmental samples were negative for any viruses or bacteria. Conclusion: Our investigation indicated that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at high-risk stalls was determined as the probable cause of the outbreak. Keywords: Foodborne, Gastroenteritis, Norovirus, Bacillus cereus, Outbreak, Cohort study Background Norovirus can be divided into at least 5 genogroups (GI–GV) and at least 35 genotypes. Human disease is primarily caused by GI and GII norovirus. GII viruses are the most frequently detected (89%), whereas GI viruses cause approximately 11% of all outbreaks [1–4]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes [5]. In the past decade, most reported norovirus outbreaks were also caused by GII.4, GI norovirus outbreaks were relatively rare, and systematic description of the epidemiology and characteristics of GI outbreaks was even rarer [6]. Yet, this outbreak was caused by the GI norovirus. Norovirus is thought to be the major cause of acute gastroenteritis [7]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last up for 1–3 days but can persist longer for young children under 5, elderly, and immunocompromised patients. The average incubation period ranges from 12 to 48 h [8]. Sporadic infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted by contact with an infected person or contaminated environmental surfaces, or by eating or drinking contaminated food or water. Per year in the United States, 31 major pathogens caused 9.4 million episodes of foodborne illness, 55,961 hospitalizations, and 1351 deaths. Most (58%) illnesses were caused by norovirus. In Australia norovirus was the leading cause of foodborne illness, accounting for 30% of illnesses caused by known pathogens [9]. While 163 norovirus outbreaks were reported in Japan, foodborne transmission accounted for 58.13% from 2001 to 2005 [10]. In foodborne norovirus outbreaks for which reported the source of contamination, 70% were caused by infected food handlers [11, 12]. On the other hand, Bacillus cereus is ubiquitous in nature, such as in plants and soil, in the enteric tract of insects and mammals. Thus, it is easily spread to food products, especially of plant origin, but is also frequently isolated from meat, eggs and dairy products [13]. Bacillus cereus causes two different types of food poisoning the diarrhoeal type and the emetic type. The diarrhoeal type of food poisoning is caused by complex enterotoxins [14, 15], produced during vegetative growth of Bacillus cereus in the small intestine [16], and incubation period ranges from 2 to 36 h [8]. While the emetic toxin is produced by growing cells in the food [13], and incubation period ranges from 8 to 16 h [8]. For both types of food poisoning the food involved has usually been heat-treated, and surviving spores are the source of the food poisoning. On September 4, 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. Totally 20 people became ill on September 4 and 5 with vomiting and diarrhoea. Food from the school canteen was considered as the source of this outbreak. Retained food specimens from the school canteen were tested positive for Bacillus cereus on September 6. Meanwhile, Stool samples obtained from 7 students were tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak just verify additional cases, source of infection, vehicle for infection, and mode of transmission. Methods Study design When the outbreak was reported, an investigation began immediately. Because of the investigation was in response to a public health emergency, and section 108 of Food Safety Law of the People’s Republic of China law (chapter 7) provides that, after a food safety incident occurs, the investigation department have the right to find out from relevant units and individuals about the situation related to the incident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus the disposal of outbreak was exempted from ethical approval and does not require informed consent. In spite of this, we still gave oral announcement to all respondents before the investigation. Ultimately we obtained a part of signed questionnaires, part of subjects by telephone interview. A questionnaire survey (see Additional file 1) includes basic personal information (name, gender, age, class, dormitory etc.), date of illness onset, duration of illness, clinical symptoms, treatment, and history of exposure to suspected food, water and patients. Moreover, we also collected the refectory records from all students and compared between cases and non-cases. All investigation data will be filed by Shunyi Center for Disease Control and Prevention, and not be disclosed to third parties. A retrospective cohort study was conducted among school students and staff to test the risk hypothesis in canteen. Canteen manager provided refectory records of 33 food stalls from August 31 to September 8. During the period from September 1 to 5, exposure to high risk foods or patients was most likely to explain the symptoms that occured from September 1 to 7 (assuming an incubation period of 2–48 h). Exposures prior to August 31 were not included in the exposure investigation as this was the summer vacation during which only few students in school. Based on daily food exposure to asymptomatic person, we established 5 crowd cohorts from September 1 to 5. Over the next 2 days, the number of postprandial cases at different stalls was divided by the total number of diners at the stall, to calculate the attack rate (AR). For each specific date (September 1, 2, 3, 4 and 5), by comparing the dining status between case and non-case in different stalls, to calculate risk ratios (RR) and 95% confidence intervals (CI). The data were inputted by EXCEL v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). pvalue was two-sided and p < 0.05 was considered statistically significant. Case definition The investigated subjects included students and staff in the university. Suspected case was defined by the onset of vomiting or diarrhoea (≥3 times per day) in the university since August 27, 2018. Laboratory confirmed case was the stool or vomit specimen of suspected case tested positive for Bacillus cereus or norovirus. Case finding We collected case information from nearby hospitals, school infirmary, and head teachers, with a special focus on patients with vomiting, abdominal pain, diarrhoea, and fever. Each case was confirmed either by face-toface questionnaire or by telephone. Laboratory and environmental investigations We collected rectal swabs, feces and vomit samples from cases, rectal swabs from canteen employees, retained food samples, and drinking water samples including tap water and self-providing well water. Detection of norovirus by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was extracted from 140 μL of fecal sample diluted 1:10 in 0.05 mol/L phosphate-buffered saline using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. RNA was stored at − 20 °C until further use. The One-Step RT-PCR Kit (QIAGEN, Hilden, Germany) was used to amplify norovirus genes in the open reading frame 1 (ORF1) and in the junction gene region between ORF1 and ORF2. Targeted to the regions B and C of the norovirus genome, a novel RT-PCR assay was performed with primers MON432/G1SKR for GI viruses and primers MON431/G2SKR for GII viruses, yielding 579 bp and 570 bp PCR products. The RT-PCR products were sent to Thermo Fisher Biochemicals (Beijing) Ltd. (Beijing, China) for sequencing using an ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli was confirmed by culture. Total bacterial count, total coliforms, thermotolerant coliforms and norovirus were detected in water samples. The culture medium was mainly provided by Beijing Land Bridge Technology Co., Ltd. (Beijing, China). Results Descriptive epidemiology We identified 209 suspected cases between August 29 and September 10, 28 of which were laboratory confirmed cases. All cases were students and age ranges from 17 to 23. The incidence rate was 4.1% (209 / 5043). The school is a boarding school, and cases were widely distributed in all 6 dormitory buildings and also in 118 classes. No aggregation of dormitories and classes was found. In addition, no cases were found among faculty members and their families, and no similar cases were found among urban residents around the school. The main clinical symptoms of the cases were nausea (70.3%), diarrhea (69.9%), vomiting (65.6%), abdominal pain (64.1%), fatigue (40.2%), dizziness (36.4%), fever (29.7%), and headache (24.9%). There were no cases of hospitalization or death. Fifty-two percent (108/209) of the cases were male, and the median age of onset was 20 years (range 17–33 years). The outbreak lasted 13 days from 17:00 on August 29. The high-peak occurred on September 5 and no further cases were reported after September 10 (Fig. 1). Analytical epidemiology In order to verify that dining in the canteen was a high risk factor, we analyzed people who ate in canteen on any day from September 1 to 5, to determine the relationship between consumption of different stalls and onset. The selected cases were those who developed the disease within 2–48 h after dining in the canteen. The incidence of daily diners from September 1 to 5 was 2.1, 1.5, 2.3, 2.4 and 1.5% over the next 2 days. Cohort study found that multiple high-risk stalls were present every day on September 1, September 2, September 4, and September 5, suggesting that those who had eaten in those stalls were at greater risk of developing the disease in the next 2 days. We didn’t find any high-risk stalls on September 3 (Table 1). Laboratory inspection Among 35 specimens of rectal swabs or feces from students, 28 specimens were positive (positive rate 80%). Norovirus GI.6 alone was detected in 23 specimens, Bacillus cereus alone in 3 specimens and both norovirus GI.6 and Bacillus cereus in 2 specimens. Rectal swabs from 124 canteen employees were tested on September 6, and 10 were positive for norovirus GI.6 (positive rate 8.1%). In addition, rectal swabs from 2 canteen employees were positive for Bacillus cereus, and one of them worked in the Barbeque stall, which a high-risk one. Of the seven retained foods, four were positive for Bacillus cereus, and the test results were in the range of 10 to 1.6 × 105 CFU/g. Norovirus was not detected in retained foods. Seven environmental samples and six drinking water samples for bacteria and norovirus tested negative. Environmental hygiene investigation Students ate only in the student canteen, which had 33 stalls. Each of the food stalls operated independently, with different types of food. On September 4, investigation found that the food raw materials and public tablewares in the kitchen operation of some stalls were disorderly, and the sanitary conditions were poor. Some of the food was stored at room temperature for a long time. The water supply mode of the school was self-built facilities water supply, the water supply sanitation license was effective, the disinfection equipment was running normally, and the water supply process met the relevant specification requirements. There were two self-supply wells in the school, no pollution sources within 30 m around the wells, and the sanitary protection of the wells accorded with the requirements. Drinking water mode was mainly drinking brand of bottled water and boiled water heated by electric water heater. According to the investigation of the canteen employees showed that 3 norovirus-positive employees reported symptoms of nausea, diarrhoea and vomiting between September 1 and 2, and continued to work until September 6. All norovirus-positive employees were closely connected, even after someone developed symptoms. They shared the toilet next to canteen, and some lived together and ate high-risk foods, such as rice flour or fish meal, made by each other (based on a survey of four people’s eating history). Eventually, their work stalls had become high-risk (such as Drinks, Rice flour and Fish meal stalls). Discussion The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. The reason for the low incidence of norovirus might be that it was limited to foodborne transmission in the short term and had not developed to the late stage of person to person transmission. Due to the appropriate measures in the later stage, norovirus had not spread completely on campus. In addition, there were only a few high-risk stalls in the canteen every day, it might also be the cause of no more cases. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost 2 weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was continuous exposure pattern rather than person to person transmission pattern. In addition, the lack of spatial aggregation of cases also indirectly supported food-borne transmission. At the same time, due to students’ resistance, early isolation of student cases was not ideal. But after the implementation of measures such as closure of high-risk stalls, isolation of canteen employees with norovirus and Bacillus cereus positive, and thorough disinfection of canteen, the outbreak quickly subsided. This further confirmed the hypothesis that food supplied in the canteen was the root cause of the outbreak. Through retrospective cohort study, daily cohort was established and personnel exposure in each stall every day was calculated. Finally, multiple high-risk stalls were found, which provided strong epidemiological evidence for food-borne transmission. Because the outbreak was continuous exposure, this approach reduced the chance that cases might be misclassified and ensured that they were placed in a cohort every day as non-sick people. Laboratory evidence suggested that the positive rate of norovirus was 71.4% (25/35) among the cases. Although this rate may be over-estimated due to testing on only some selected cases, it was still much higher than the prevalence of norovirus reported in patients (17%) with acute gastroenteritis in developing countries [17], supporting the causative role of norovirus in this outbreak. Moreover, our study is the first reported outbreak of genotype GI.6 norovirus in the Shunyi District of Beijing, China. Compared with non-GI.6 outbreak, the GI.6 outbreak was characterized by food-borne transmission [18]. When canteen employees served in these high-risk stalls, food might be contaminated by their faeces, or unhygienic practices by employees who expelled viruses and bacteria. Viruses and bacteria could also be excreted without symptoms, so improper handling by asymptomatic food handlers could also lead to outbreaks. Bacillus cereus had also been detected in the outbreak, which is believed to be an opportunistic pathogen that causes gastrointestinal symptoms associated with the production of cereulide (emetic toxin) or enterotoxin (diarrheal syndrome). In this outbreak, between 10 and 1.6 × 105 CFU/g Bacillus cereus were detected in the incriminated foods, and diarrheal or emetic syndrome was often associated with Bacillus cereus counts of 105 to 108 cells or spores [19]. In addition, Bacillus cereus was detected in stool samples of canteen employees and cases, and one of employees who tested positive sold Bacillus cereus-positive foods. Based on the experience of the outbreak, the following suggestions are put forward. Firstly, preventive measures such as cases for Isolation, health education, cleaning and disinfection of dwelling and dining place, identification and exclusion of symptomatic food handlers, even if the cause of the disease is not clear, should be carried out at an early stage, to avoid further development of the outbreak. Secondly, epidemiological investigations need to be carried out in a timely manner, especially in this outbreak caused by norovirus, transmission of the virus is diverse, and it is very difficult to detect the virus in contaminated food because of the low titer of virus. So, to identify risk factors, it is all the more important to use epidemiological investigation. Finally, in order to quickly identify pathogens that cause for outbreaks, samples still need to be submitted at the same time for pathogen detection, and to implement targeted control measures. There were also some limitations in this study. Firstly, it was a large-scale outbreak, but the number of investigators was limited. This had led to some cases not being verified and underestimating the incidence of the population. Secondly, only the existence of high-risk stalls was investigated, and the information on food manufacture and sale could not be obtained. As a result, this made it impossible to analyze key control links in the processing and storage of food. Finally, some canteen employees might have failed to reveal any symptoms of gastroenteritis to the investigation team due to fear of adverse consequences, but this information was important to further trace the source. Conclusion Our survey showed that canteen employees were infected by two pathogens (norovirus and Bacillus cereus) and transmission may have been possible due to unhygienic practices. Student consumption of food or drink at highrisk stalls was determined as the probable cause of the outbreak. Epidemiological investigation proved to be useful in determining the probable source of the infection and to implement timely intervention measures.

What were the infrastructure complaints?

194

A large and persistent outbreak of typhoid fever caused by consuming contaminated water and street-vended beverages Kampala, Uganda, January

Abstract Background: On 6 February 2015, Kampala city authorities alerted the Ugandan Ministry of Health of a “strange disease†that killed one person and sickened dozens. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods: We defined a suspected case as onset of fever (≥37.5 °C) for more than 3 days with abdominal pain, headache, negative malaria test or failed anti-malaria treatment, and at least 2 of the following: diarrhea, nausea or vomiting, constipation, fatigue. A probable case was defined as a suspected case with a positive TUBEX® TF test. A confirmed case had blood culture yielding Salmonella Typhi. We conducted a case-control study to compare exposures of 33 suspected case-patients and 78 controls, and tested water and juice samples. Results: From 17 February-12 June, we identified 10,230 suspected, 1038 probable, and 51 confirmed cases. Approximately 22.58% (7/31) of case-patients and 2.56% (2/78) of controls drank water sold in small plastic bags (ORM-H = 8.90; 95%CI = 1.60–49.00); 54.54% (18/33) of case-patients and 19.23% (15/78) of controls consumed locallymade drinks (ORM-H = 4.60; 95%CI: 1.90–11.00). All isolates were susceptible to ciprofloxacin and ceftriaxone. Water and juice samples exhibited evidence of fecal contamination. Conclusion: Contaminated water and street-vended beverages were likely vehicles of this outbreak. At our recommendation authorities closed unsafe water sources and supplied safe water to affected areas. Keywords: Typhoid fever, Outbreak, Case-control, Uganda Background Typhoid fever is a systemic disease caused by Salmonella enterica serovar Typhi, a Gram-negative bacterium. Humans are the only host, and transmission most commonly occurs through ingestion of water or food contaminated by feces from an acutely ill or convalescent patient or an asymptomatic carrier. The incubation period is usually 1 to 2 weeks but can range from 3 to 60 days [1]. The illness presents with sustained fever and a constellation of other symptoms including dry cough, fatigue, abdominal pain, diarrhea, and constipation [2]. Case fatality ratios range between 10 and 30% if untreated, but fall to 1–4% with appropriate and timely antimicrobial treatment [3]. The gold standard laboratory diagnosis of typhoid fever requires isolation of S.. Typhi from blood, stool, bone marrow, or other tissue or bodily fluid by bacterial culture [2]. Other tests with moderate sensitivity and specificity include the Widal test and TUBEX® TF test which involve detection of antibodies against S. Typhi antigens [2]. Typhoid fever is preventable through public health interventions such as provision of safe water, ensuring proper sanitation and waste disposal systems, and excluding disease carriers from handling food [4]. Typhoid fever is a major cause of mortality and morbidity worldwide. In endemic areas, the disease is most commonly found in children 5–19 years of age. International visitors from non-endemic areas are also at risk if unvaccinated [1]. The global burden of the disease in low- and middle-income countries in 2010 was estimated to be 11.9 million cases, including 129,000 fatalities, after adjusting for water-related risk factors [5]. In Uganda, an outbreak of typhoid fever in Kasese District sickened 8092 persons from 27 December 2007 to 30 July 2009, resulting in at least 249 intestinal perforations and 47 deaths [6]. In 2011, numerous typhoid cases were again reported in Kasese and neighboring Bundibugyo District with many more intestinal perforations and emergence of multidrug resistant strains [7]. On 6 February, 2015, the Ugandan Ministry of Health (MoH) received a report from the Kampala Capital City Authority that a 42-year-old man had died a day earlier of a “strange illness.†The patient was admitted to the hospital on 2 February 2015 with symptoms of abdominal pain, high fever, and severe jaundice. Initial testing involved use of the Widal test which was positive. Approximately 30 other people who worked in the same area as the deceased reportedly had similar symptoms. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods Study sites The outbreak occurred in Kampala (estimated population 1.4 million), the capital of Uganda [8]. Kampala has five divisions: Kampala Central, Kawempe, Makindye, Rubaga, and Nakawa. We focused our epidemiologic investigation on two markets and a commuter taxi park in Kampala Central Division where the initial cases were concentrated. Surveillance To characterize and control the epidemic, MoH conducted surveillance at six treatment centers established in affected areas of the city to provide diagnostic testing and typhoid fever treatment free of charge. These treatment centers were existing health centers in which routine disease surveillance and treatment activities are conducted. Through the media, local leaders encouraged the people with symptoms of typhoid fever to seek medical care at these treatment centers. We defined a suspected case as onset of fever (≥37.5 °C) for ≥3 days from 1 January 2015 onwards, with headache, abdominal pain, a negative test for malaria or failure to respond to anti-malaria treatment, and ≥2 of the following symptoms: diarrhea, nausea or vomiting, constipation, or fatigue. A probable case was a suspected case whose serum sample yielded a positive TUBEX® TF test [9]. Blood samples were collected from the first 5 suspected cases every day from each treatment center and referred to the microbiology laboratory at the Medical Research Council for blood culture. A confirmed case was a suspected case whose blood culture yielded S. Typhi. Case-control study We conducted open-ended hypothesis-generating interviews of case-patients found in the areas where the initial cases were identified, focusing on their usual sources of water and food. To test the hypotheses generated from the interviews, we conducted a case-control study from 10 to 20 February 2015. To rapidly identify the mode of transmission so as to inform prompt prevention and control measures, we used the initial 33 suspected case-patients identified in the earliest-affected communities for the case-control study. The earliest cases were persons working in two markets or in the central terminal station for Kampala’s shared taxis, all of which were located in central Kampala. Therefore we recruited both the cases and the controls from those places. The markets are open spaces where people set up their stalls to sell assorted merchandise, whereas the central terminal station for the shared taxis is an area where the shared taxis (mini-vans) pick up and drop off passengers. In the markets, after identifying and interviewing a case, the interviewer then walked around the stall to identify several persons of the same gender and similar age as the case from the surrounding stalls who never had a febrile disease since January 1, 2015, and recruited those persons as controls. Similarly, in the central terminal station for the shared taxis, after identifying and interviewing a case who was working inside a shared taxi (e.g., a driver or conductor), the interviewer then walked around the shared taxi to recruit asymptomatic workers of the same gender and similar age from the surrounding shared taxis as controls. The interviewers used a structured questionnaire to collect information on the usual water and food exposures from the case- and control-persons. A link to the questionnaire that was used has been provided in the Additional file 1 of the manuscript. Clinical laboratory investigation The TUBEX® TF test was performed at the treatment centers by trained clinical and laboratory staff as per the manufacturer’s instructions. Blood culture was performed on the first five patients presenting each day at the 6 treatment centers. From each adult patient, 5–10 mL of blood was collected and inoculated in BD Bactecâ„¢ Aerobic/F blood culture bottles and incubated in a BD Bactec 9000 seriesâ„¢. Presumptive positive bottles, as signaled by the system, were subcultured on MacConkey, chocolate, and blood agar plates and incubated aerobically at 37 °C for 24 h. A Gram stain was also performed. Negative vials were incubated for up to 7 days and if the system still indicated negative, a Gram stain was performed and a final subculture was done before reporting the specimen as negative. Oxidase-negative, lactose non-fermenting colonies, were screened using API 10S at the start of the outbreak. Later an abbreviated panel of biochemical tests [10] was used. Isolates biochemically typical of S. Typhi were serotyped using slide agglutination with S. polyvalent O, S. polyvalent H, S. O factor 9 (group D), S. H factor d and S. Vi antisera. A set of 30 S. Typhi isolates were sent to the U.S. Centers for Disease Control and Prevention (CDC) for confirmation and antimicrobial susceptibility testing (AST). The National Antimicrobial Resistance Monitoring System at CDC performed AST on 17 isolates by broth microdilution to determine minimum inhibitory concentrations for 14 antimicrobial agents: amoxicillin/clavulanic acid, ampicillin, azithromycin, ceftiofur, ceftriaxone, cefoxitin, chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Results were interpreted using Clinical and Laboratory Standards Institute standards [11] when available. During the case-control study, we collected 5– 10 mL of blood from each of 20 suspected casepatients, placed the samples into commercial BD Bactecâ„¢ Aerobic/F media, and transported them to the clinical laboratory at the Makerere College of Health Sciences Department of Medical Microbiology for incubation in the Bactec 9120â„¢ blood culture system. Subcultures onto MacConkey and blood agar were done following instrument signals of growth or at the end of 7 days of incubation. Colonies were identified as S. Typhi based on biochemical characteristics including motility, hydrogen sulfide production, fermentation of sugars, urease production, and serological typing characteristics with various specific antisera. Environmental laboratory investigation From 2 to 8 April, juice samples were collected from the Nakasero, Owino, and Shauriyako markets, and 100 mL water samples were collected from unprotected water sources such as unprotected springs (i.e., underground water sources that do not have barriers protecting them from contamination and run-off) and commercial vendors in Kampala Central Division. We chose these water collection sites because we observed people in the outbreak-affected areas collecting water from these sites. The juice samples were tested because case-persons said they usually consumed these drinks. We collected nine juice samples, including 3 “bushera†(millet and yeast), 2 “munanansi†(pineapple juice with tea leaves), 3 “butunda†(passion fruit), and 1 “bongo†(unpasteurized yogurt drink). We also collected 13 water samples, including 3 “kaveera†(water packaged and sold in a small plastic bag), one unlabeled bottle of water from a street vendor, water from three storage tanks, and water from five unprotected springs. Juice and water samples were tested using a modified version of the United States Environmental Protection Agency’s Standard Analytical Protocol for S. Typhi in Drinking Water [12]. Briefly, 125 mL of specimen was preenriched in 125 mL of double strength buffered peptone water at 37 °C, followed by parallel enrichment in Selenite Cysteine broth at 37 °C and RV broth at 42 °C. Cultures from Selenite Cysteine broth were plated onto MacConkey and XLD agars; cultures from RV broth were plated onto XLD agar. All plates were incubated at 37 °C. Plates were inspected at 24 and 48 h for colony morphology consistent with Enterobacteriacea. Colonies morphologically consistent with S. spp. (i.e. lactose negative) were subjected to biochemical testing. Suspect isolates were sent to CDC-Atlanta for biochemical confirmation. For confirmation, suspect cultures were streaked onto Hektoen enteric agar and suspect colonies were subjected to an abbreviated panel of tests, for phenotypic identification of Salmonella or Shigella spp. and biochemical differentiation of S. serovars Typhi and Paratyphi A from other Salmonella serovars [10]. Statistical analysis Using surveillance data, the attack rates by sex, division, and sex were calculated using population data from the national census [8] and data provided by the Uganda Bureau of Statistics [13]. Using the StatCalc in Epi Info 7, considering a power of 80%, two sided confidence level of 95%, a case-control ratio of 1:2 with 30% of cases exposed and 10% of controls exposed, we would require about 39 cases and 77 controls. To measure the associations between exposure variables and illness status, we used the Mantel-Haenszel method to estimate odds ratios (OR) and their confidence intervals, accounting for frequency-matching of cases and controls. We calculated the proportion of cases and controls who drank 1, 2, and 3–4 types of locally made drinks, and used the Chisquare test for linear trend to assess the relationship between the number of types of drinks consumed and odds of illness [14]. Results Surveillance From 17 February to 12 June 2015, we identified 10,230 suspected cases from the six treatment centers established by MoH. Cases were distributed widely throughout Kampala and neighboring areas (Fig. 1). The epidemic curve of suspected cases suggests that the outbreak started at the beginning of February or perhaps earlier. By the time the outbreak was recognized on 6 February, hundreds of cases had already occurred (Fig. 2). Cases were reported in all five divisions of Kampala: Makindye (32%, 3234), Rubaga (28%, 2828), Kawempe (11%, 1144), Nakawa (6.4%, 656) and Central (4.2%, 428); for 19% (1940) of cases, either no division of residence was identified, or resided outside of Kampala. The attack rate during the outbreak period was highest in Makindye (10/1,000), Rubaga (8.7/1000), and Central (6.5/1000) Divisions. Males had a higher attack rate than females. The attack rate among people in the 15–59 year age group (12/1000) was 6 times higher than among younger (2.0/1000) or older (2.0/1000) persons (Table 1). Case-control study In our hypothesis-generating interviews of patients from the area where the outbreak was first identified, consumption of drinks made with water extracted from unprotected sources and packed in unhygienic conditions was often reported. Of the 33 case-patients we enrolled in the case-control study, 60% were men; the majority of the case-patients (85%) were in the 20–39 year and 9.1% in older age groups. In addition to fever, commonly reported symptoms included abdominal pain (72.72%) and headache (69.69%) (Table 2). We found that 22.58% (7/31) of case patients compared with 2.56% (2/78) of controls usually drank locally packaged water in small plastic bags called “kaveera water†(ORM-H = 8.90; 95% CI = 1.60–49.00); 55% (18/33) of case-patients compared with 19.23% (15/78) of controls drank locally-made passion fruit juice called “butunda†(ORM-H = 4.60; 95% CI: 1.90– 11.00); 31.25% (10/32) of case-patients compared with 16.67% (13/78) of controls usually drank locally-packed pineapple juice called “munanansi†(ORM-H = 2.00; 95% CI = 0.74–5.20); and 15.63% (5/32) of case-patients compared with 8.97% (7/78) of controls usually drank cold millet porridge called “bushera†(ORM-H = 2.80; 95% CI = 0.76–10.00). Workplace as a source of breakfast (ORM-H = 0.25; 95% CI = 0.07–0.93), and workplace as a source of lunch (ORM-H = 0.35; 95% CI = 0.11–1.10) were not significant risk factors for illness. When we compared the proportions of case-patients and controls who drank 0, 1, 2, or 3–4 types of locallymade drinks, we found that case-patients were more likely to drink multiple types of locally-made drinks than controls (Chi-square for linear trend = 14.65, p < 0.001) (Table 3). Laboratory investigation Of 10,230 suspected cases, 3464 (10%) underwent TUBEX® TF testing. Of those, 1038 were positive, representing a positivity rate of 29%. Blood samples from a total of 364 patients (including 20 of 33 case-control study patients) were tested by blood culture and 56 (15%) (including 5 of the 20 case-control study patients tested) yielded S. enterica ser. Typhi. Subsequently, 30 of the 56 S. Typhi isolates from blood cultures were confirmed at the U.S. CDC as S. Typhi. CDC determined the minimum inhibitory concentrations for 17 of these isolates, 5 of which were resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, nalidixic acid, trimethoprim/sulfamethoxazole and had intermediate interpretation to ciprofloxacin. The remaining 12 were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. Environmental investigation One of 3 “kaveera water†samples and the unlabeled bottle of water sold by street vendors contained lactose fermenting bacteria, which are commonly Enterobacteriaceae and associated with fecal contamination. The 2 other “kaveera water†samples contained non-lactose fermenting bacteria, also consistent with fecal contamination, and one isolate was further identified as non-typhoidal Salmonella. The 5 water samples from unprotected springs showed evidence of robust contamination with lactose fermenting bacteria. Lactose non-fermenting colonies from 2 spring water samples were identified as non-typhoidal Salmonella spp. Lactose fermenting bacteria were also detected in 2 of 3 “bushera†samples, 1 of 2 “munanasi†samples, all 3 “munanansi†juice samples, and the “bongo†sample. Additionally, non-typhoidal Salmonella was cultured from 1 “busheraâ€, 1 “munanansiâ€, and 1 passion fruit juice sample. Discussion Our investigation revealed a prolonged and widespread outbreak of typhoid fever that affected thousands of people in all five divisions of Kampala City over several months. Contaminated water from unprotected sources and drinks made with it were the likely vehicles of infection early in the outbreak. Juice and water samples obtained from street vendors and water samples collected from unprotected spring water sources showed evidence of fecal contamination. Although S. Typhi was not recovered from environmental testing, non-Typhi Salmonella were isolated from five street-vended beverage samples from the implicated markets. All 17 isolates of S. Typhi from blood tested at CDC were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. It is possible that persons affected by the antibiotic-resistant strains during this outbreak had experienced complications of typhoid fever considering that antibiotic-resistant strains of S. Typhi are associated with more severe form of the illness, complications and death [15]. This outbreak may have started in January 2015 or even earlier; however, it was not recognized until early February because routine clinical and laboratory surveillance systems for typhoid fever were not in place before the investigation. The widespread nature of the outbreak is compatible with a waterborne source. The sudden increase in cases after the start of the investigation was likely due to active community outreach and education about the symptoms of typhoid fever and the availability of prompt, free diagnostic testing and treatment through newly established treatment centers. The gradual decline in cases from mid-March onwards was likely the result of patient treatment and public health interventions including provision of free water chlorination products, sensitization of residents on water treatment, and the establishment of free alternative safe water sources in the most affected communities. Based on the evidence we presented, the Kampala Capital City Authority sealed off all underground water sources and worked with the National Water and Sewerage Corporation to ensure the provision of accessible alternative sources of water to the affected communities. In Uganda, as in many low and middle income countries, definitive diagnostic tests for typhoid fever such as blood culture are usually unavailable, unaffordable, or inconsistently applied [16]. Instead, typhoid fever diagnosis and surveillance often rely on clinical judgment or on the Widal test, which has poor sensitivity and specificity [17]. Moreover, physicians often give presumptive antibiotic and/or antimalarial treatment for febrile illnesses [18, 19] without attempting to determine the etiology. Previous studies have indicated that a significant proportion of febrile illness in Uganda is caused by bacteremia, including invasive non-Typhi salmonellosis and typhoid fever [20]. A more robust approach in these settings could entail periodically identifying persons with febrile illness in the communities and taking blood culture for confirmation [21]. The blood samples could be collected and sent using a specialized transport network to regional laboratory centers around the country where confirmative tests can be performed. This system has been successfully used to improve diagnostic services in early infant HIV/AIDS diagnosis [22]. Sentinel surveillance for febrile illnesses based on blood cultures would accelerate the early identification of outbreaks and implementation of control measures. Waterborne typhoid and paratyphoid fever affect an estimated 27 million people worldwide each year [3]. In developing countries, where safe water and sanitation systems have not been well-established, large-scale typhoid and paratyphoid outbreaks sometimes occur [7, 23–26]. During a previous typhoid outbreak in Kasese and Bundibugyo districts, Uganda, in 2009–2011, which affected 8092 persons, the vehicle of transmission was also found to be unclean water [7]. The current outbreak was likely caused by consuming contaminated water from unprotected ground water sources. Kampala city has more than 200 unprotected ground water sources, most of which serve as unprotected sources of water for economically disadvantaged people in the city such as those in our investigation [27]. Unsafe disposal of excreta and solid waste are significant factors that contribute to contamination of ground water in Kampala [28]. This outbreak investigation highlights the importance of ensuring access to affordable, safe, treated drinking water and improved sanitation and waste management systems for resource-constrained urban populations. Risk factors for typhoid transmission were not assessed later during this outbreak, when foodborne transmission might have become more common. Recurrent contamination of unprotected water sources with S. Typhi likely continued to sustain the outbreak propagation over the course of several months. According to the Uganda Demographic Health Survey 2011 [29], almost 30% of people living in urban areas and more than 60% of those living in rural areas do not treat their water before drinking it. Barriers to safer drinking water include the cost associated with establishing a piped treated water system or purchasing water treatment products for household use and the false perception that naturally occurring water sources could be safe [30]. In the aftermath of outbreaks like this one, public health authorities face 3 possible options: The first option is to do nothing but respond to outbreaks as they occur. Governments in resource-constrained settings often choose this option, leaving the population vulnerable to outbreaks of waterborne diseases including cholera, hepatitis A and E, cryptosporidiosis, shigellosis, and many others in addition to typhoid and paratyphoid fever. The second option is mass vaccination against typhoid fever. A cost-effectiveness evaluation of a hypothetical typhoid vaccination campaign was carried out after the multi-year outbreak of typhoid fever in Kasese District, Uganda, and it was estimated to be highly costeffective [31]. However, vaccination against the many different pathogens that cause waterborne diseases is not possible because vaccines are not yet available for many of them (e.g. cryptosporidiosis, shigellosis, paratyphoid fever, etc.). In addition, typhoid fever vaccines have been shown to have varied levels of effectiveness (from 50 to 95%) and to last for varied lengths of time (from 3 to 10 years) [15]. The third and final option is to improve the water and sanitation systems. Improvement of sanitation, hygiene and clean water supply around the world could avert ≥90% of diarrheal disease episodes annually [32]. In North America and Europe, typhoid fever caused largescale outbreaks from the late 19th through the early 20th century [33, 34]. After improvement of municipal water and sanitation systems in the early 20th century, waterborne outbreaks of communicable diseases including typhoid fever drastically decreased [35–37]. Improved sanitation measures such as having a basic pit latrine or a toilet connected to a septic tank curtail the direct contact between human waste and water or the environment. Yet in 2012, only 33% of the urban population in Uganda had access to adequate sanitation, an increase of only 1% since 1990, and 2% still practiced open defecation [38]. Although improving water and sanitation systems requires a substantial investment by the government, ultimately it is highly cost-effective in the reduction of many waterborne diseases [39]. Strengths and limitations A major limitation of our investigation was that, due to inadequate laboratory capacity to confirm a large number of cases early in the outbreak, and the need to rapidly identify the mode of transmission to inform effective interventions, we included non-laboratory confirmed cases in our case-control study. While a clinical case definition for typhoid fever cases can lead to misclassification, and is not recommended during nonoutbreak situations, during an outbreak such a case definition will often perform well, as measured by good positive and negative predictive values [40]. Also, the information on usual sources of water and food was based on self-reports, which could represent a source ofinformation bias. Another limitation is that data on mortality or on complications such as intestinal perforations were not collected. With over 10,000 cases it is likely that there were intestinal perforations and deaths but no surveillance for those outcomes was done. A study is currently being conducted to assess these severe impacts of this outbreak. In addition, only a few water and juice samples were tested, which could explain why S. Typhi was not isolated during the environmental investigation. Also, water and juice samples were tested using the reagents and procedures that were available in the laboratory for testing clinical specimens for Salmonella, and not more conventional methods for evaluating the potential presence of fecal contamination in these types of samples. Conclusion In conclusion, this investigation revealed a large outbreak of typhoid fever that affected thousands of people in Kampala, Uganda, which appeared to have been caused by consuming contaminated water and local drinks made from it. To prevent future waterborne outbreaks, we recommended that the Kampala Capital City Authority, the MoH, the National Water and Sewerage Corporation, and partners invest in improving access to potable water, and safe sanitation and hygiene facilities [41].

What happened?

195

A large and persistent outbreak of typhoid fever caused by consuming contaminated water and street-vended beverages Kampala, Uganda, January

Abstract Background: On 6 February 2015, Kampala city authorities alerted the Ugandan Ministry of Health of a “strange disease†that killed one person and sickened dozens. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods: We defined a suspected case as onset of fever (≥37.5 °C) for more than 3 days with abdominal pain, headache, negative malaria test or failed anti-malaria treatment, and at least 2 of the following: diarrhea, nausea or vomiting, constipation, fatigue. A probable case was defined as a suspected case with a positive TUBEX® TF test. A confirmed case had blood culture yielding Salmonella Typhi. We conducted a case-control study to compare exposures of 33 suspected case-patients and 78 controls, and tested water and juice samples. Results: From 17 February-12 June, we identified 10,230 suspected, 1038 probable, and 51 confirmed cases. Approximately 22.58% (7/31) of case-patients and 2.56% (2/78) of controls drank water sold in small plastic bags (ORM-H = 8.90; 95%CI = 1.60–49.00); 54.54% (18/33) of case-patients and 19.23% (15/78) of controls consumed locallymade drinks (ORM-H = 4.60; 95%CI: 1.90–11.00). All isolates were susceptible to ciprofloxacin and ceftriaxone. Water and juice samples exhibited evidence of fecal contamination. Conclusion: Contaminated water and street-vended beverages were likely vehicles of this outbreak. At our recommendation authorities closed unsafe water sources and supplied safe water to affected areas. Keywords: Typhoid fever, Outbreak, Case-control, Uganda Background Typhoid fever is a systemic disease caused by Salmonella enterica serovar Typhi, a Gram-negative bacterium. Humans are the only host, and transmission most commonly occurs through ingestion of water or food contaminated by feces from an acutely ill or convalescent patient or an asymptomatic carrier. The incubation period is usually 1 to 2 weeks but can range from 3 to 60 days [1]. The illness presents with sustained fever and a constellation of other symptoms including dry cough, fatigue, abdominal pain, diarrhea, and constipation [2]. Case fatality ratios range between 10 and 30% if untreated, but fall to 1–4% with appropriate and timely antimicrobial treatment [3]. The gold standard laboratory diagnosis of typhoid fever requires isolation of S.. Typhi from blood, stool, bone marrow, or other tissue or bodily fluid by bacterial culture [2]. Other tests with moderate sensitivity and specificity include the Widal test and TUBEX® TF test which involve detection of antibodies against S. Typhi antigens [2]. Typhoid fever is preventable through public health interventions such as provision of safe water, ensuring proper sanitation and waste disposal systems, and excluding disease carriers from handling food [4]. Typhoid fever is a major cause of mortality and morbidity worldwide. In endemic areas, the disease is most commonly found in children 5–19 years of age. International visitors from non-endemic areas are also at risk if unvaccinated [1]. The global burden of the disease in low- and middle-income countries in 2010 was estimated to be 11.9 million cases, including 129,000 fatalities, after adjusting for water-related risk factors [5]. In Uganda, an outbreak of typhoid fever in Kasese District sickened 8092 persons from 27 December 2007 to 30 July 2009, resulting in at least 249 intestinal perforations and 47 deaths [6]. In 2011, numerous typhoid cases were again reported in Kasese and neighboring Bundibugyo District with many more intestinal perforations and emergence of multidrug resistant strains [7]. On 6 February, 2015, the Ugandan Ministry of Health (MoH) received a report from the Kampala Capital City Authority that a 42-year-old man had died a day earlier of a “strange illness.†The patient was admitted to the hospital on 2 February 2015 with symptoms of abdominal pain, high fever, and severe jaundice. Initial testing involved use of the Widal test which was positive. Approximately 30 other people who worked in the same area as the deceased reportedly had similar symptoms. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods Study sites The outbreak occurred in Kampala (estimated population 1.4 million), the capital of Uganda [8]. Kampala has five divisions: Kampala Central, Kawempe, Makindye, Rubaga, and Nakawa. We focused our epidemiologic investigation on two markets and a commuter taxi park in Kampala Central Division where the initial cases were concentrated. Surveillance To characterize and control the epidemic, MoH conducted surveillance at six treatment centers established in affected areas of the city to provide diagnostic testing and typhoid fever treatment free of charge. These treatment centers were existing health centers in which routine disease surveillance and treatment activities are conducted. Through the media, local leaders encouraged the people with symptoms of typhoid fever to seek medical care at these treatment centers. We defined a suspected case as onset of fever (≥37.5 °C) for ≥3 days from 1 January 2015 onwards, with headache, abdominal pain, a negative test for malaria or failure to respond to anti-malaria treatment, and ≥2 of the following symptoms: diarrhea, nausea or vomiting, constipation, or fatigue. A probable case was a suspected case whose serum sample yielded a positive TUBEX® TF test [9]. Blood samples were collected from the first 5 suspected cases every day from each treatment center and referred to the microbiology laboratory at the Medical Research Council for blood culture. A confirmed case was a suspected case whose blood culture yielded S. Typhi. Case-control study We conducted open-ended hypothesis-generating interviews of case-patients found in the areas where the initial cases were identified, focusing on their usual sources of water and food. To test the hypotheses generated from the interviews, we conducted a case-control study from 10 to 20 February 2015. To rapidly identify the mode of transmission so as to inform prompt prevention and control measures, we used the initial 33 suspected case-patients identified in the earliest-affected communities for the case-control study. The earliest cases were persons working in two markets or in the central terminal station for Kampala’s shared taxis, all of which were located in central Kampala. Therefore we recruited both the cases and the controls from those places. The markets are open spaces where people set up their stalls to sell assorted merchandise, whereas the central terminal station for the shared taxis is an area where the shared taxis (mini-vans) pick up and drop off passengers. In the markets, after identifying and interviewing a case, the interviewer then walked around the stall to identify several persons of the same gender and similar age as the case from the surrounding stalls who never had a febrile disease since January 1, 2015, and recruited those persons as controls. Similarly, in the central terminal station for the shared taxis, after identifying and interviewing a case who was working inside a shared taxi (e.g., a driver or conductor), the interviewer then walked around the shared taxi to recruit asymptomatic workers of the same gender and similar age from the surrounding shared taxis as controls. The interviewers used a structured questionnaire to collect information on the usual water and food exposures from the case- and control-persons. A link to the questionnaire that was used has been provided in the Additional file 1 of the manuscript. Clinical laboratory investigation The TUBEX® TF test was performed at the treatment centers by trained clinical and laboratory staff as per the manufacturer’s instructions. Blood culture was performed on the first five patients presenting each day at the 6 treatment centers. From each adult patient, 5–10 mL of blood was collected and inoculated in BD Bactecâ„¢ Aerobic/F blood culture bottles and incubated in a BD Bactec 9000 seriesâ„¢. Presumptive positive bottles, as signaled by the system, were subcultured on MacConkey, chocolate, and blood agar plates and incubated aerobically at 37 °C for 24 h. A Gram stain was also performed. Negative vials were incubated for up to 7 days and if the system still indicated negative, a Gram stain was performed and a final subculture was done before reporting the specimen as negative. Oxidase-negative, lactose non-fermenting colonies, were screened using API 10S at the start of the outbreak. Later an abbreviated panel of biochemical tests [10] was used. Isolates biochemically typical of S. Typhi were serotyped using slide agglutination with S. polyvalent O, S. polyvalent H, S. O factor 9 (group D), S. H factor d and S. Vi antisera. A set of 30 S. Typhi isolates were sent to the U.S. Centers for Disease Control and Prevention (CDC) for confirmation and antimicrobial susceptibility testing (AST). The National Antimicrobial Resistance Monitoring System at CDC performed AST on 17 isolates by broth microdilution to determine minimum inhibitory concentrations for 14 antimicrobial agents: amoxicillin/clavulanic acid, ampicillin, azithromycin, ceftiofur, ceftriaxone, cefoxitin, chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Results were interpreted using Clinical and Laboratory Standards Institute standards [11] when available. During the case-control study, we collected 5– 10 mL of blood from each of 20 suspected casepatients, placed the samples into commercial BD Bactecâ„¢ Aerobic/F media, and transported them to the clinical laboratory at the Makerere College of Health Sciences Department of Medical Microbiology for incubation in the Bactec 9120â„¢ blood culture system. Subcultures onto MacConkey and blood agar were done following instrument signals of growth or at the end of 7 days of incubation. Colonies were identified as S. Typhi based on biochemical characteristics including motility, hydrogen sulfide production, fermentation of sugars, urease production, and serological typing characteristics with various specific antisera. Environmental laboratory investigation From 2 to 8 April, juice samples were collected from the Nakasero, Owino, and Shauriyako markets, and 100 mL water samples were collected from unprotected water sources such as unprotected springs (i.e., underground water sources that do not have barriers protecting them from contamination and run-off) and commercial vendors in Kampala Central Division. We chose these water collection sites because we observed people in the outbreak-affected areas collecting water from these sites. The juice samples were tested because case-persons said they usually consumed these drinks. We collected nine juice samples, including 3 “bushera†(millet and yeast), 2 “munanansi†(pineapple juice with tea leaves), 3 “butunda†(passion fruit), and 1 “bongo†(unpasteurized yogurt drink). We also collected 13 water samples, including 3 “kaveera†(water packaged and sold in a small plastic bag), one unlabeled bottle of water from a street vendor, water from three storage tanks, and water from five unprotected springs. Juice and water samples were tested using a modified version of the United States Environmental Protection Agency’s Standard Analytical Protocol for S. Typhi in Drinking Water [12]. Briefly, 125 mL of specimen was preenriched in 125 mL of double strength buffered peptone water at 37 °C, followed by parallel enrichment in Selenite Cysteine broth at 37 °C and RV broth at 42 °C. Cultures from Selenite Cysteine broth were plated onto MacConkey and XLD agars; cultures from RV broth were plated onto XLD agar. All plates were incubated at 37 °C. Plates were inspected at 24 and 48 h for colony morphology consistent with Enterobacteriacea. Colonies morphologically consistent with S. spp. (i.e. lactose negative) were subjected to biochemical testing. Suspect isolates were sent to CDC-Atlanta for biochemical confirmation. For confirmation, suspect cultures were streaked onto Hektoen enteric agar and suspect colonies were subjected to an abbreviated panel of tests, for phenotypic identification of Salmonella or Shigella spp. and biochemical differentiation of S. serovars Typhi and Paratyphi A from other Salmonella serovars [10]. Statistical analysis Using surveillance data, the attack rates by sex, division, and sex were calculated using population data from the national census [8] and data provided by the Uganda Bureau of Statistics [13]. Using the StatCalc in Epi Info 7, considering a power of 80%, two sided confidence level of 95%, a case-control ratio of 1:2 with 30% of cases exposed and 10% of controls exposed, we would require about 39 cases and 77 controls. To measure the associations between exposure variables and illness status, we used the Mantel-Haenszel method to estimate odds ratios (OR) and their confidence intervals, accounting for frequency-matching of cases and controls. We calculated the proportion of cases and controls who drank 1, 2, and 3–4 types of locally made drinks, and used the Chisquare test for linear trend to assess the relationship between the number of types of drinks consumed and odds of illness [14]. Results Surveillance From 17 February to 12 June 2015, we identified 10,230 suspected cases from the six treatment centers established by MoH. Cases were distributed widely throughout Kampala and neighboring areas (Fig. 1). The epidemic curve of suspected cases suggests that the outbreak started at the beginning of February or perhaps earlier. By the time the outbreak was recognized on 6 February, hundreds of cases had already occurred (Fig. 2). Cases were reported in all five divisions of Kampala: Makindye (32%, 3234), Rubaga (28%, 2828), Kawempe (11%, 1144), Nakawa (6.4%, 656) and Central (4.2%, 428); for 19% (1940) of cases, either no division of residence was identified, or resided outside of Kampala. The attack rate during the outbreak period was highest in Makindye (10/1,000), Rubaga (8.7/1000), and Central (6.5/1000) Divisions. Males had a higher attack rate than females. The attack rate among people in the 15–59 year age group (12/1000) was 6 times higher than among younger (2.0/1000) or older (2.0/1000) persons (Table 1). Case-control study In our hypothesis-generating interviews of patients from the area where the outbreak was first identified, consumption of drinks made with water extracted from unprotected sources and packed in unhygienic conditions was often reported. Of the 33 case-patients we enrolled in the case-control study, 60% were men; the majority of the case-patients (85%) were in the 20–39 year and 9.1% in older age groups. In addition to fever, commonly reported symptoms included abdominal pain (72.72%) and headache (69.69%) (Table 2). We found that 22.58% (7/31) of case patients compared with 2.56% (2/78) of controls usually drank locally packaged water in small plastic bags called “kaveera water†(ORM-H = 8.90; 95% CI = 1.60–49.00); 55% (18/33) of case-patients compared with 19.23% (15/78) of controls drank locally-made passion fruit juice called “butunda†(ORM-H = 4.60; 95% CI: 1.90– 11.00); 31.25% (10/32) of case-patients compared with 16.67% (13/78) of controls usually drank locally-packed pineapple juice called “munanansi†(ORM-H = 2.00; 95% CI = 0.74–5.20); and 15.63% (5/32) of case-patients compared with 8.97% (7/78) of controls usually drank cold millet porridge called “bushera†(ORM-H = 2.80; 95% CI = 0.76–10.00). Workplace as a source of breakfast (ORM-H = 0.25; 95% CI = 0.07–0.93), and workplace as a source of lunch (ORM-H = 0.35; 95% CI = 0.11–1.10) were not significant risk factors for illness. When we compared the proportions of case-patients and controls who drank 0, 1, 2, or 3–4 types of locallymade drinks, we found that case-patients were more likely to drink multiple types of locally-made drinks than controls (Chi-square for linear trend = 14.65, p < 0.001) (Table 3). Laboratory investigation Of 10,230 suspected cases, 3464 (10%) underwent TUBEX® TF testing. Of those, 1038 were positive, representing a positivity rate of 29%. Blood samples from a total of 364 patients (including 20 of 33 case-control study patients) were tested by blood culture and 56 (15%) (including 5 of the 20 case-control study patients tested) yielded S. enterica ser. Typhi. Subsequently, 30 of the 56 S. Typhi isolates from blood cultures were confirmed at the U.S. CDC as S. Typhi. CDC determined the minimum inhibitory concentrations for 17 of these isolates, 5 of which were resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, nalidixic acid, trimethoprim/sulfamethoxazole and had intermediate interpretation to ciprofloxacin. The remaining 12 were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. Environmental investigation One of 3 “kaveera water†samples and the unlabeled bottle of water sold by street vendors contained lactose fermenting bacteria, which are commonly Enterobacteriaceae and associated with fecal contamination. The 2 other “kaveera water†samples contained non-lactose fermenting bacteria, also consistent with fecal contamination, and one isolate was further identified as non-typhoidal Salmonella. The 5 water samples from unprotected springs showed evidence of robust contamination with lactose fermenting bacteria. Lactose non-fermenting colonies from 2 spring water samples were identified as non-typhoidal Salmonella spp. Lactose fermenting bacteria were also detected in 2 of 3 “bushera†samples, 1 of 2 “munanasi†samples, all 3 “munanansi†juice samples, and the “bongo†sample. Additionally, non-typhoidal Salmonella was cultured from 1 “busheraâ€, 1 “munanansiâ€, and 1 passion fruit juice sample. Discussion Our investigation revealed a prolonged and widespread outbreak of typhoid fever that affected thousands of people in all five divisions of Kampala City over several months. Contaminated water from unprotected sources and drinks made with it were the likely vehicles of infection early in the outbreak. Juice and water samples obtained from street vendors and water samples collected from unprotected spring water sources showed evidence of fecal contamination. Although S. Typhi was not recovered from environmental testing, non-Typhi Salmonella were isolated from five street-vended beverage samples from the implicated markets. All 17 isolates of S. Typhi from blood tested at CDC were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. It is possible that persons affected by the antibiotic-resistant strains during this outbreak had experienced complications of typhoid fever considering that antibiotic-resistant strains of S. Typhi are associated with more severe form of the illness, complications and death [15]. This outbreak may have started in January 2015 or even earlier; however, it was not recognized until early February because routine clinical and laboratory surveillance systems for typhoid fever were not in place before the investigation. The widespread nature of the outbreak is compatible with a waterborne source. The sudden increase in cases after the start of the investigation was likely due to active community outreach and education about the symptoms of typhoid fever and the availability of prompt, free diagnostic testing and treatment through newly established treatment centers. The gradual decline in cases from mid-March onwards was likely the result of patient treatment and public health interventions including provision of free water chlorination products, sensitization of residents on water treatment, and the establishment of free alternative safe water sources in the most affected communities. Based on the evidence we presented, the Kampala Capital City Authority sealed off all underground water sources and worked with the National Water and Sewerage Corporation to ensure the provision of accessible alternative sources of water to the affected communities. In Uganda, as in many low and middle income countries, definitive diagnostic tests for typhoid fever such as blood culture are usually unavailable, unaffordable, or inconsistently applied [16]. Instead, typhoid fever diagnosis and surveillance often rely on clinical judgment or on the Widal test, which has poor sensitivity and specificity [17]. Moreover, physicians often give presumptive antibiotic and/or antimalarial treatment for febrile illnesses [18, 19] without attempting to determine the etiology. Previous studies have indicated that a significant proportion of febrile illness in Uganda is caused by bacteremia, including invasive non-Typhi salmonellosis and typhoid fever [20]. A more robust approach in these settings could entail periodically identifying persons with febrile illness in the communities and taking blood culture for confirmation [21]. The blood samples could be collected and sent using a specialized transport network to regional laboratory centers around the country where confirmative tests can be performed. This system has been successfully used to improve diagnostic services in early infant HIV/AIDS diagnosis [22]. Sentinel surveillance for febrile illnesses based on blood cultures would accelerate the early identification of outbreaks and implementation of control measures. Waterborne typhoid and paratyphoid fever affect an estimated 27 million people worldwide each year [3]. In developing countries, where safe water and sanitation systems have not been well-established, large-scale typhoid and paratyphoid outbreaks sometimes occur [7, 23–26]. During a previous typhoid outbreak in Kasese and Bundibugyo districts, Uganda, in 2009–2011, which affected 8092 persons, the vehicle of transmission was also found to be unclean water [7]. The current outbreak was likely caused by consuming contaminated water from unprotected ground water sources. Kampala city has more than 200 unprotected ground water sources, most of which serve as unprotected sources of water for economically disadvantaged people in the city such as those in our investigation [27]. Unsafe disposal of excreta and solid waste are significant factors that contribute to contamination of ground water in Kampala [28]. This outbreak investigation highlights the importance of ensuring access to affordable, safe, treated drinking water and improved sanitation and waste management systems for resource-constrained urban populations. Risk factors for typhoid transmission were not assessed later during this outbreak, when foodborne transmission might have become more common. Recurrent contamination of unprotected water sources with S. Typhi likely continued to sustain the outbreak propagation over the course of several months. According to the Uganda Demographic Health Survey 2011 [29], almost 30% of people living in urban areas and more than 60% of those living in rural areas do not treat their water before drinking it. Barriers to safer drinking water include the cost associated with establishing a piped treated water system or purchasing water treatment products for household use and the false perception that naturally occurring water sources could be safe [30]. In the aftermath of outbreaks like this one, public health authorities face 3 possible options: The first option is to do nothing but respond to outbreaks as they occur. Governments in resource-constrained settings often choose this option, leaving the population vulnerable to outbreaks of waterborne diseases including cholera, hepatitis A and E, cryptosporidiosis, shigellosis, and many others in addition to typhoid and paratyphoid fever. The second option is mass vaccination against typhoid fever. A cost-effectiveness evaluation of a hypothetical typhoid vaccination campaign was carried out after the multi-year outbreak of typhoid fever in Kasese District, Uganda, and it was estimated to be highly costeffective [31]. However, vaccination against the many different pathogens that cause waterborne diseases is not possible because vaccines are not yet available for many of them (e.g. cryptosporidiosis, shigellosis, paratyphoid fever, etc.). In addition, typhoid fever vaccines have been shown to have varied levels of effectiveness (from 50 to 95%) and to last for varied lengths of time (from 3 to 10 years) [15]. The third and final option is to improve the water and sanitation systems. Improvement of sanitation, hygiene and clean water supply around the world could avert ≥90% of diarrheal disease episodes annually [32]. In North America and Europe, typhoid fever caused largescale outbreaks from the late 19th through the early 20th century [33, 34]. After improvement of municipal water and sanitation systems in the early 20th century, waterborne outbreaks of communicable diseases including typhoid fever drastically decreased [35–37]. Improved sanitation measures such as having a basic pit latrine or a toilet connected to a septic tank curtail the direct contact between human waste and water or the environment. Yet in 2012, only 33% of the urban population in Uganda had access to adequate sanitation, an increase of only 1% since 1990, and 2% still practiced open defecation [38]. Although improving water and sanitation systems requires a substantial investment by the government, ultimately it is highly cost-effective in the reduction of many waterborne diseases [39]. Strengths and limitations A major limitation of our investigation was that, due to inadequate laboratory capacity to confirm a large number of cases early in the outbreak, and the need to rapidly identify the mode of transmission to inform effective interventions, we included non-laboratory confirmed cases in our case-control study. While a clinical case definition for typhoid fever cases can lead to misclassification, and is not recommended during nonoutbreak situations, during an outbreak such a case definition will often perform well, as measured by good positive and negative predictive values [40]. Also, the information on usual sources of water and food was based on self-reports, which could represent a source ofinformation bias. Another limitation is that data on mortality or on complications such as intestinal perforations were not collected. With over 10,000 cases it is likely that there were intestinal perforations and deaths but no surveillance for those outcomes was done. A study is currently being conducted to assess these severe impacts of this outbreak. In addition, only a few water and juice samples were tested, which could explain why S. Typhi was not isolated during the environmental investigation. Also, water and juice samples were tested using the reagents and procedures that were available in the laboratory for testing clinical specimens for Salmonella, and not more conventional methods for evaluating the potential presence of fecal contamination in these types of samples. Conclusion In conclusion, this investigation revealed a large outbreak of typhoid fever that affected thousands of people in Kampala, Uganda, which appeared to have been caused by consuming contaminated water and local drinks made from it. To prevent future waterborne outbreaks, we recommended that the Kampala Capital City Authority, the MoH, the National Water and Sewerage Corporation, and partners invest in improving access to potable water, and safe sanitation and hygiene facilities [41].

What was the event?

196

A large and persistent outbreak of typhoid fever caused by consuming contaminated water and street-vended beverages Kampala, Uganda, January

Abstract Background: On 6 February 2015, Kampala city authorities alerted the Ugandan Ministry of Health of a “strange disease†that killed one person and sickened dozens. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods: We defined a suspected case as onset of fever (≥37.5 °C) for more than 3 days with abdominal pain, headache, negative malaria test or failed anti-malaria treatment, and at least 2 of the following: diarrhea, nausea or vomiting, constipation, fatigue. A probable case was defined as a suspected case with a positive TUBEX® TF test. A confirmed case had blood culture yielding Salmonella Typhi. We conducted a case-control study to compare exposures of 33 suspected case-patients and 78 controls, and tested water and juice samples. Results: From 17 February-12 June, we identified 10,230 suspected, 1038 probable, and 51 confirmed cases. Approximately 22.58% (7/31) of case-patients and 2.56% (2/78) of controls drank water sold in small plastic bags (ORM-H = 8.90; 95%CI = 1.60–49.00); 54.54% (18/33) of case-patients and 19.23% (15/78) of controls consumed locallymade drinks (ORM-H = 4.60; 95%CI: 1.90–11.00). All isolates were susceptible to ciprofloxacin and ceftriaxone. Water and juice samples exhibited evidence of fecal contamination. Conclusion: Contaminated water and street-vended beverages were likely vehicles of this outbreak. At our recommendation authorities closed unsafe water sources and supplied safe water to affected areas. Keywords: Typhoid fever, Outbreak, Case-control, Uganda Background Typhoid fever is a systemic disease caused by Salmonella enterica serovar Typhi, a Gram-negative bacterium. Humans are the only host, and transmission most commonly occurs through ingestion of water or food contaminated by feces from an acutely ill or convalescent patient or an asymptomatic carrier. The incubation period is usually 1 to 2 weeks but can range from 3 to 60 days [1]. The illness presents with sustained fever and a constellation of other symptoms including dry cough, fatigue, abdominal pain, diarrhea, and constipation [2]. Case fatality ratios range between 10 and 30% if untreated, but fall to 1–4% with appropriate and timely antimicrobial treatment [3]. The gold standard laboratory diagnosis of typhoid fever requires isolation of S.. Typhi from blood, stool, bone marrow, or other tissue or bodily fluid by bacterial culture [2]. Other tests with moderate sensitivity and specificity include the Widal test and TUBEX® TF test which involve detection of antibodies against S. Typhi antigens [2]. Typhoid fever is preventable through public health interventions such as provision of safe water, ensuring proper sanitation and waste disposal systems, and excluding disease carriers from handling food [4]. Typhoid fever is a major cause of mortality and morbidity worldwide. In endemic areas, the disease is most commonly found in children 5–19 years of age. International visitors from non-endemic areas are also at risk if unvaccinated [1]. The global burden of the disease in low- and middle-income countries in 2010 was estimated to be 11.9 million cases, including 129,000 fatalities, after adjusting for water-related risk factors [5]. In Uganda, an outbreak of typhoid fever in Kasese District sickened 8092 persons from 27 December 2007 to 30 July 2009, resulting in at least 249 intestinal perforations and 47 deaths [6]. In 2011, numerous typhoid cases were again reported in Kasese and neighboring Bundibugyo District with many more intestinal perforations and emergence of multidrug resistant strains [7]. On 6 February, 2015, the Ugandan Ministry of Health (MoH) received a report from the Kampala Capital City Authority that a 42-year-old man had died a day earlier of a “strange illness.†The patient was admitted to the hospital on 2 February 2015 with symptoms of abdominal pain, high fever, and severe jaundice. Initial testing involved use of the Widal test which was positive. Approximately 30 other people who worked in the same area as the deceased reportedly had similar symptoms. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods Study sites The outbreak occurred in Kampala (estimated population 1.4 million), the capital of Uganda [8]. Kampala has five divisions: Kampala Central, Kawempe, Makindye, Rubaga, and Nakawa. We focused our epidemiologic investigation on two markets and a commuter taxi park in Kampala Central Division where the initial cases were concentrated. Surveillance To characterize and control the epidemic, MoH conducted surveillance at six treatment centers established in affected areas of the city to provide diagnostic testing and typhoid fever treatment free of charge. These treatment centers were existing health centers in which routine disease surveillance and treatment activities are conducted. Through the media, local leaders encouraged the people with symptoms of typhoid fever to seek medical care at these treatment centers. We defined a suspected case as onset of fever (≥37.5 °C) for ≥3 days from 1 January 2015 onwards, with headache, abdominal pain, a negative test for malaria or failure to respond to anti-malaria treatment, and ≥2 of the following symptoms: diarrhea, nausea or vomiting, constipation, or fatigue. A probable case was a suspected case whose serum sample yielded a positive TUBEX® TF test [9]. Blood samples were collected from the first 5 suspected cases every day from each treatment center and referred to the microbiology laboratory at the Medical Research Council for blood culture. A confirmed case was a suspected case whose blood culture yielded S. Typhi. Case-control study We conducted open-ended hypothesis-generating interviews of case-patients found in the areas where the initial cases were identified, focusing on their usual sources of water and food. To test the hypotheses generated from the interviews, we conducted a case-control study from 10 to 20 February 2015. To rapidly identify the mode of transmission so as to inform prompt prevention and control measures, we used the initial 33 suspected case-patients identified in the earliest-affected communities for the case-control study. The earliest cases were persons working in two markets or in the central terminal station for Kampala’s shared taxis, all of which were located in central Kampala. Therefore we recruited both the cases and the controls from those places. The markets are open spaces where people set up their stalls to sell assorted merchandise, whereas the central terminal station for the shared taxis is an area where the shared taxis (mini-vans) pick up and drop off passengers. In the markets, after identifying and interviewing a case, the interviewer then walked around the stall to identify several persons of the same gender and similar age as the case from the surrounding stalls who never had a febrile disease since January 1, 2015, and recruited those persons as controls. Similarly, in the central terminal station for the shared taxis, after identifying and interviewing a case who was working inside a shared taxi (e.g., a driver or conductor), the interviewer then walked around the shared taxi to recruit asymptomatic workers of the same gender and similar age from the surrounding shared taxis as controls. The interviewers used a structured questionnaire to collect information on the usual water and food exposures from the case- and control-persons. A link to the questionnaire that was used has been provided in the Additional file 1 of the manuscript. Clinical laboratory investigation The TUBEX® TF test was performed at the treatment centers by trained clinical and laboratory staff as per the manufacturer’s instructions. Blood culture was performed on the first five patients presenting each day at the 6 treatment centers. From each adult patient, 5–10 mL of blood was collected and inoculated in BD Bactecâ„¢ Aerobic/F blood culture bottles and incubated in a BD Bactec 9000 seriesâ„¢. Presumptive positive bottles, as signaled by the system, were subcultured on MacConkey, chocolate, and blood agar plates and incubated aerobically at 37 °C for 24 h. A Gram stain was also performed. Negative vials were incubated for up to 7 days and if the system still indicated negative, a Gram stain was performed and a final subculture was done before reporting the specimen as negative. Oxidase-negative, lactose non-fermenting colonies, were screened using API 10S at the start of the outbreak. Later an abbreviated panel of biochemical tests [10] was used. Isolates biochemically typical of S. Typhi were serotyped using slide agglutination with S. polyvalent O, S. polyvalent H, S. O factor 9 (group D), S. H factor d and S. Vi antisera. A set of 30 S. Typhi isolates were sent to the U.S. Centers for Disease Control and Prevention (CDC) for confirmation and antimicrobial susceptibility testing (AST). The National Antimicrobial Resistance Monitoring System at CDC performed AST on 17 isolates by broth microdilution to determine minimum inhibitory concentrations for 14 antimicrobial agents: amoxicillin/clavulanic acid, ampicillin, azithromycin, ceftiofur, ceftriaxone, cefoxitin, chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Results were interpreted using Clinical and Laboratory Standards Institute standards [11] when available. During the case-control study, we collected 5– 10 mL of blood from each of 20 suspected casepatients, placed the samples into commercial BD Bactecâ„¢ Aerobic/F media, and transported them to the clinical laboratory at the Makerere College of Health Sciences Department of Medical Microbiology for incubation in the Bactec 9120â„¢ blood culture system. Subcultures onto MacConkey and blood agar were done following instrument signals of growth or at the end of 7 days of incubation. Colonies were identified as S. Typhi based on biochemical characteristics including motility, hydrogen sulfide production, fermentation of sugars, urease production, and serological typing characteristics with various specific antisera. Environmental laboratory investigation From 2 to 8 April, juice samples were collected from the Nakasero, Owino, and Shauriyako markets, and 100 mL water samples were collected from unprotected water sources such as unprotected springs (i.e., underground water sources that do not have barriers protecting them from contamination and run-off) and commercial vendors in Kampala Central Division. We chose these water collection sites because we observed people in the outbreak-affected areas collecting water from these sites. The juice samples were tested because case-persons said they usually consumed these drinks. We collected nine juice samples, including 3 “bushera†(millet and yeast), 2 “munanansi†(pineapple juice with tea leaves), 3 “butunda†(passion fruit), and 1 “bongo†(unpasteurized yogurt drink). We also collected 13 water samples, including 3 “kaveera†(water packaged and sold in a small plastic bag), one unlabeled bottle of water from a street vendor, water from three storage tanks, and water from five unprotected springs. Juice and water samples were tested using a modified version of the United States Environmental Protection Agency’s Standard Analytical Protocol for S. Typhi in Drinking Water [12]. Briefly, 125 mL of specimen was preenriched in 125 mL of double strength buffered peptone water at 37 °C, followed by parallel enrichment in Selenite Cysteine broth at 37 °C and RV broth at 42 °C. Cultures from Selenite Cysteine broth were plated onto MacConkey and XLD agars; cultures from RV broth were plated onto XLD agar. All plates were incubated at 37 °C. Plates were inspected at 24 and 48 h for colony morphology consistent with Enterobacteriacea. Colonies morphologically consistent with S. spp. (i.e. lactose negative) were subjected to biochemical testing. Suspect isolates were sent to CDC-Atlanta for biochemical confirmation. For confirmation, suspect cultures were streaked onto Hektoen enteric agar and suspect colonies were subjected to an abbreviated panel of tests, for phenotypic identification of Salmonella or Shigella spp. and biochemical differentiation of S. serovars Typhi and Paratyphi A from other Salmonella serovars [10]. Statistical analysis Using surveillance data, the attack rates by sex, division, and sex were calculated using population data from the national census [8] and data provided by the Uganda Bureau of Statistics [13]. Using the StatCalc in Epi Info 7, considering a power of 80%, two sided confidence level of 95%, a case-control ratio of 1:2 with 30% of cases exposed and 10% of controls exposed, we would require about 39 cases and 77 controls. To measure the associations between exposure variables and illness status, we used the Mantel-Haenszel method to estimate odds ratios (OR) and their confidence intervals, accounting for frequency-matching of cases and controls. We calculated the proportion of cases and controls who drank 1, 2, and 3–4 types of locally made drinks, and used the Chisquare test for linear trend to assess the relationship between the number of types of drinks consumed and odds of illness [14]. Results Surveillance From 17 February to 12 June 2015, we identified 10,230 suspected cases from the six treatment centers established by MoH. Cases were distributed widely throughout Kampala and neighboring areas (Fig. 1). The epidemic curve of suspected cases suggests that the outbreak started at the beginning of February or perhaps earlier. By the time the outbreak was recognized on 6 February, hundreds of cases had already occurred (Fig. 2). Cases were reported in all five divisions of Kampala: Makindye (32%, 3234), Rubaga (28%, 2828), Kawempe (11%, 1144), Nakawa (6.4%, 656) and Central (4.2%, 428); for 19% (1940) of cases, either no division of residence was identified, or resided outside of Kampala. The attack rate during the outbreak period was highest in Makindye (10/1,000), Rubaga (8.7/1000), and Central (6.5/1000) Divisions. Males had a higher attack rate than females. The attack rate among people in the 15–59 year age group (12/1000) was 6 times higher than among younger (2.0/1000) or older (2.0/1000) persons (Table 1). Case-control study In our hypothesis-generating interviews of patients from the area where the outbreak was first identified, consumption of drinks made with water extracted from unprotected sources and packed in unhygienic conditions was often reported. Of the 33 case-patients we enrolled in the case-control study, 60% were men; the majority of the case-patients (85%) were in the 20–39 year and 9.1% in older age groups. In addition to fever, commonly reported symptoms included abdominal pain (72.72%) and headache (69.69%) (Table 2). We found that 22.58% (7/31) of case patients compared with 2.56% (2/78) of controls usually drank locally packaged water in small plastic bags called “kaveera water†(ORM-H = 8.90; 95% CI = 1.60–49.00); 55% (18/33) of case-patients compared with 19.23% (15/78) of controls drank locally-made passion fruit juice called “butunda†(ORM-H = 4.60; 95% CI: 1.90– 11.00); 31.25% (10/32) of case-patients compared with 16.67% (13/78) of controls usually drank locally-packed pineapple juice called “munanansi†(ORM-H = 2.00; 95% CI = 0.74–5.20); and 15.63% (5/32) of case-patients compared with 8.97% (7/78) of controls usually drank cold millet porridge called “bushera†(ORM-H = 2.80; 95% CI = 0.76–10.00). Workplace as a source of breakfast (ORM-H = 0.25; 95% CI = 0.07–0.93), and workplace as a source of lunch (ORM-H = 0.35; 95% CI = 0.11–1.10) were not significant risk factors for illness. When we compared the proportions of case-patients and controls who drank 0, 1, 2, or 3–4 types of locallymade drinks, we found that case-patients were more likely to drink multiple types of locally-made drinks than controls (Chi-square for linear trend = 14.65, p < 0.001) (Table 3). Laboratory investigation Of 10,230 suspected cases, 3464 (10%) underwent TUBEX® TF testing. Of those, 1038 were positive, representing a positivity rate of 29%. Blood samples from a total of 364 patients (including 20 of 33 case-control study patients) were tested by blood culture and 56 (15%) (including 5 of the 20 case-control study patients tested) yielded S. enterica ser. Typhi. Subsequently, 30 of the 56 S. Typhi isolates from blood cultures were confirmed at the U.S. CDC as S. Typhi. CDC determined the minimum inhibitory concentrations for 17 of these isolates, 5 of which were resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, nalidixic acid, trimethoprim/sulfamethoxazole and had intermediate interpretation to ciprofloxacin. The remaining 12 were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. Environmental investigation One of 3 “kaveera water†samples and the unlabeled bottle of water sold by street vendors contained lactose fermenting bacteria, which are commonly Enterobacteriaceae and associated with fecal contamination. The 2 other “kaveera water†samples contained non-lactose fermenting bacteria, also consistent with fecal contamination, and one isolate was further identified as non-typhoidal Salmonella. The 5 water samples from unprotected springs showed evidence of robust contamination with lactose fermenting bacteria. Lactose non-fermenting colonies from 2 spring water samples were identified as non-typhoidal Salmonella spp. Lactose fermenting bacteria were also detected in 2 of 3 “bushera†samples, 1 of 2 “munanasi†samples, all 3 “munanansi†juice samples, and the “bongo†sample. Additionally, non-typhoidal Salmonella was cultured from 1 “busheraâ€, 1 “munanansiâ€, and 1 passion fruit juice sample. Discussion Our investigation revealed a prolonged and widespread outbreak of typhoid fever that affected thousands of people in all five divisions of Kampala City over several months. Contaminated water from unprotected sources and drinks made with it were the likely vehicles of infection early in the outbreak. Juice and water samples obtained from street vendors and water samples collected from unprotected spring water sources showed evidence of fecal contamination. Although S. Typhi was not recovered from environmental testing, non-Typhi Salmonella were isolated from five street-vended beverage samples from the implicated markets. All 17 isolates of S. Typhi from blood tested at CDC were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. It is possible that persons affected by the antibiotic-resistant strains during this outbreak had experienced complications of typhoid fever considering that antibiotic-resistant strains of S. Typhi are associated with more severe form of the illness, complications and death [15]. This outbreak may have started in January 2015 or even earlier; however, it was not recognized until early February because routine clinical and laboratory surveillance systems for typhoid fever were not in place before the investigation. The widespread nature of the outbreak is compatible with a waterborne source. The sudden increase in cases after the start of the investigation was likely due to active community outreach and education about the symptoms of typhoid fever and the availability of prompt, free diagnostic testing and treatment through newly established treatment centers. The gradual decline in cases from mid-March onwards was likely the result of patient treatment and public health interventions including provision of free water chlorination products, sensitization of residents on water treatment, and the establishment of free alternative safe water sources in the most affected communities. Based on the evidence we presented, the Kampala Capital City Authority sealed off all underground water sources and worked with the National Water and Sewerage Corporation to ensure the provision of accessible alternative sources of water to the affected communities. In Uganda, as in many low and middle income countries, definitive diagnostic tests for typhoid fever such as blood culture are usually unavailable, unaffordable, or inconsistently applied [16]. Instead, typhoid fever diagnosis and surveillance often rely on clinical judgment or on the Widal test, which has poor sensitivity and specificity [17]. Moreover, physicians often give presumptive antibiotic and/or antimalarial treatment for febrile illnesses [18, 19] without attempting to determine the etiology. Previous studies have indicated that a significant proportion of febrile illness in Uganda is caused by bacteremia, including invasive non-Typhi salmonellosis and typhoid fever [20]. A more robust approach in these settings could entail periodically identifying persons with febrile illness in the communities and taking blood culture for confirmation [21]. The blood samples could be collected and sent using a specialized transport network to regional laboratory centers around the country where confirmative tests can be performed. This system has been successfully used to improve diagnostic services in early infant HIV/AIDS diagnosis [22]. Sentinel surveillance for febrile illnesses based on blood cultures would accelerate the early identification of outbreaks and implementation of control measures. Waterborne typhoid and paratyphoid fever affect an estimated 27 million people worldwide each year [3]. In developing countries, where safe water and sanitation systems have not been well-established, large-scale typhoid and paratyphoid outbreaks sometimes occur [7, 23–26]. During a previous typhoid outbreak in Kasese and Bundibugyo districts, Uganda, in 2009–2011, which affected 8092 persons, the vehicle of transmission was also found to be unclean water [7]. The current outbreak was likely caused by consuming contaminated water from unprotected ground water sources. Kampala city has more than 200 unprotected ground water sources, most of which serve as unprotected sources of water for economically disadvantaged people in the city such as those in our investigation [27]. Unsafe disposal of excreta and solid waste are significant factors that contribute to contamination of ground water in Kampala [28]. This outbreak investigation highlights the importance of ensuring access to affordable, safe, treated drinking water and improved sanitation and waste management systems for resource-constrained urban populations. Risk factors for typhoid transmission were not assessed later during this outbreak, when foodborne transmission might have become more common. Recurrent contamination of unprotected water sources with S. Typhi likely continued to sustain the outbreak propagation over the course of several months. According to the Uganda Demographic Health Survey 2011 [29], almost 30% of people living in urban areas and more than 60% of those living in rural areas do not treat their water before drinking it. Barriers to safer drinking water include the cost associated with establishing a piped treated water system or purchasing water treatment products for household use and the false perception that naturally occurring water sources could be safe [30]. In the aftermath of outbreaks like this one, public health authorities face 3 possible options: The first option is to do nothing but respond to outbreaks as they occur. Governments in resource-constrained settings often choose this option, leaving the population vulnerable to outbreaks of waterborne diseases including cholera, hepatitis A and E, cryptosporidiosis, shigellosis, and many others in addition to typhoid and paratyphoid fever. The second option is mass vaccination against typhoid fever. A cost-effectiveness evaluation of a hypothetical typhoid vaccination campaign was carried out after the multi-year outbreak of typhoid fever in Kasese District, Uganda, and it was estimated to be highly costeffective [31]. However, vaccination against the many different pathogens that cause waterborne diseases is not possible because vaccines are not yet available for many of them (e.g. cryptosporidiosis, shigellosis, paratyphoid fever, etc.). In addition, typhoid fever vaccines have been shown to have varied levels of effectiveness (from 50 to 95%) and to last for varied lengths of time (from 3 to 10 years) [15]. The third and final option is to improve the water and sanitation systems. Improvement of sanitation, hygiene and clean water supply around the world could avert ≥90% of diarrheal disease episodes annually [32]. In North America and Europe, typhoid fever caused largescale outbreaks from the late 19th through the early 20th century [33, 34]. After improvement of municipal water and sanitation systems in the early 20th century, waterborne outbreaks of communicable diseases including typhoid fever drastically decreased [35–37]. Improved sanitation measures such as having a basic pit latrine or a toilet connected to a septic tank curtail the direct contact between human waste and water or the environment. Yet in 2012, only 33% of the urban population in Uganda had access to adequate sanitation, an increase of only 1% since 1990, and 2% still practiced open defecation [38]. Although improving water and sanitation systems requires a substantial investment by the government, ultimately it is highly cost-effective in the reduction of many waterborne diseases [39]. Strengths and limitations A major limitation of our investigation was that, due to inadequate laboratory capacity to confirm a large number of cases early in the outbreak, and the need to rapidly identify the mode of transmission to inform effective interventions, we included non-laboratory confirmed cases in our case-control study. While a clinical case definition for typhoid fever cases can lead to misclassification, and is not recommended during nonoutbreak situations, during an outbreak such a case definition will often perform well, as measured by good positive and negative predictive values [40]. Also, the information on usual sources of water and food was based on self-reports, which could represent a source ofinformation bias. Another limitation is that data on mortality or on complications such as intestinal perforations were not collected. With over 10,000 cases it is likely that there were intestinal perforations and deaths but no surveillance for those outcomes was done. A study is currently being conducted to assess these severe impacts of this outbreak. In addition, only a few water and juice samples were tested, which could explain why S. Typhi was not isolated during the environmental investigation. Also, water and juice samples were tested using the reagents and procedures that were available in the laboratory for testing clinical specimens for Salmonella, and not more conventional methods for evaluating the potential presence of fecal contamination in these types of samples. Conclusion In conclusion, this investigation revealed a large outbreak of typhoid fever that affected thousands of people in Kampala, Uganda, which appeared to have been caused by consuming contaminated water and local drinks made from it. To prevent future waterborne outbreaks, we recommended that the Kampala Capital City Authority, the MoH, the National Water and Sewerage Corporation, and partners invest in improving access to potable water, and safe sanitation and hygiene facilities [41].

When did this happen?

197

A large and persistent outbreak of typhoid fever caused by consuming contaminated water and street-vended beverages Kampala, Uganda, January

Abstract Background: On 6 February 2015, Kampala city authorities alerted the Ugandan Ministry of Health of a “strange disease†that killed one person and sickened dozens. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods: We defined a suspected case as onset of fever (≥37.5 °C) for more than 3 days with abdominal pain, headache, negative malaria test or failed anti-malaria treatment, and at least 2 of the following: diarrhea, nausea or vomiting, constipation, fatigue. A probable case was defined as a suspected case with a positive TUBEX® TF test. A confirmed case had blood culture yielding Salmonella Typhi. We conducted a case-control study to compare exposures of 33 suspected case-patients and 78 controls, and tested water and juice samples. Results: From 17 February-12 June, we identified 10,230 suspected, 1038 probable, and 51 confirmed cases. Approximately 22.58% (7/31) of case-patients and 2.56% (2/78) of controls drank water sold in small plastic bags (ORM-H = 8.90; 95%CI = 1.60–49.00); 54.54% (18/33) of case-patients and 19.23% (15/78) of controls consumed locallymade drinks (ORM-H = 4.60; 95%CI: 1.90–11.00). All isolates were susceptible to ciprofloxacin and ceftriaxone. Water and juice samples exhibited evidence of fecal contamination. Conclusion: Contaminated water and street-vended beverages were likely vehicles of this outbreak. At our recommendation authorities closed unsafe water sources and supplied safe water to affected areas. Keywords: Typhoid fever, Outbreak, Case-control, Uganda Background Typhoid fever is a systemic disease caused by Salmonella enterica serovar Typhi, a Gram-negative bacterium. Humans are the only host, and transmission most commonly occurs through ingestion of water or food contaminated by feces from an acutely ill or convalescent patient or an asymptomatic carrier. The incubation period is usually 1 to 2 weeks but can range from 3 to 60 days [1]. The illness presents with sustained fever and a constellation of other symptoms including dry cough, fatigue, abdominal pain, diarrhea, and constipation [2]. Case fatality ratios range between 10 and 30% if untreated, but fall to 1–4% with appropriate and timely antimicrobial treatment [3]. The gold standard laboratory diagnosis of typhoid fever requires isolation of S.. Typhi from blood, stool, bone marrow, or other tissue or bodily fluid by bacterial culture [2]. Other tests with moderate sensitivity and specificity include the Widal test and TUBEX® TF test which involve detection of antibodies against S. Typhi antigens [2]. Typhoid fever is preventable through public health interventions such as provision of safe water, ensuring proper sanitation and waste disposal systems, and excluding disease carriers from handling food [4]. Typhoid fever is a major cause of mortality and morbidity worldwide. In endemic areas, the disease is most commonly found in children 5–19 years of age. International visitors from non-endemic areas are also at risk if unvaccinated [1]. The global burden of the disease in low- and middle-income countries in 2010 was estimated to be 11.9 million cases, including 129,000 fatalities, after adjusting for water-related risk factors [5]. In Uganda, an outbreak of typhoid fever in Kasese District sickened 8092 persons from 27 December 2007 to 30 July 2009, resulting in at least 249 intestinal perforations and 47 deaths [6]. In 2011, numerous typhoid cases were again reported in Kasese and neighboring Bundibugyo District with many more intestinal perforations and emergence of multidrug resistant strains [7]. On 6 February, 2015, the Ugandan Ministry of Health (MoH) received a report from the Kampala Capital City Authority that a 42-year-old man had died a day earlier of a “strange illness.†The patient was admitted to the hospital on 2 February 2015 with symptoms of abdominal pain, high fever, and severe jaundice. Initial testing involved use of the Widal test which was positive. Approximately 30 other people who worked in the same area as the deceased reportedly had similar symptoms. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods Study sites The outbreak occurred in Kampala (estimated population 1.4 million), the capital of Uganda [8]. Kampala has five divisions: Kampala Central, Kawempe, Makindye, Rubaga, and Nakawa. We focused our epidemiologic investigation on two markets and a commuter taxi park in Kampala Central Division where the initial cases were concentrated. Surveillance To characterize and control the epidemic, MoH conducted surveillance at six treatment centers established in affected areas of the city to provide diagnostic testing and typhoid fever treatment free of charge. These treatment centers were existing health centers in which routine disease surveillance and treatment activities are conducted. Through the media, local leaders encouraged the people with symptoms of typhoid fever to seek medical care at these treatment centers. We defined a suspected case as onset of fever (≥37.5 °C) for ≥3 days from 1 January 2015 onwards, with headache, abdominal pain, a negative test for malaria or failure to respond to anti-malaria treatment, and ≥2 of the following symptoms: diarrhea, nausea or vomiting, constipation, or fatigue. A probable case was a suspected case whose serum sample yielded a positive TUBEX® TF test [9]. Blood samples were collected from the first 5 suspected cases every day from each treatment center and referred to the microbiology laboratory at the Medical Research Council for blood culture. A confirmed case was a suspected case whose blood culture yielded S. Typhi. Case-control study We conducted open-ended hypothesis-generating interviews of case-patients found in the areas where the initial cases were identified, focusing on their usual sources of water and food. To test the hypotheses generated from the interviews, we conducted a case-control study from 10 to 20 February 2015. To rapidly identify the mode of transmission so as to inform prompt prevention and control measures, we used the initial 33 suspected case-patients identified in the earliest-affected communities for the case-control study. The earliest cases were persons working in two markets or in the central terminal station for Kampala’s shared taxis, all of which were located in central Kampala. Therefore we recruited both the cases and the controls from those places. The markets are open spaces where people set up their stalls to sell assorted merchandise, whereas the central terminal station for the shared taxis is an area where the shared taxis (mini-vans) pick up and drop off passengers. In the markets, after identifying and interviewing a case, the interviewer then walked around the stall to identify several persons of the same gender and similar age as the case from the surrounding stalls who never had a febrile disease since January 1, 2015, and recruited those persons as controls. Similarly, in the central terminal station for the shared taxis, after identifying and interviewing a case who was working inside a shared taxi (e.g., a driver or conductor), the interviewer then walked around the shared taxi to recruit asymptomatic workers of the same gender and similar age from the surrounding shared taxis as controls. The interviewers used a structured questionnaire to collect information on the usual water and food exposures from the case- and control-persons. A link to the questionnaire that was used has been provided in the Additional file 1 of the manuscript. Clinical laboratory investigation The TUBEX® TF test was performed at the treatment centers by trained clinical and laboratory staff as per the manufacturer’s instructions. Blood culture was performed on the first five patients presenting each day at the 6 treatment centers. From each adult patient, 5–10 mL of blood was collected and inoculated in BD Bactecâ„¢ Aerobic/F blood culture bottles and incubated in a BD Bactec 9000 seriesâ„¢. Presumptive positive bottles, as signaled by the system, were subcultured on MacConkey, chocolate, and blood agar plates and incubated aerobically at 37 °C for 24 h. A Gram stain was also performed. Negative vials were incubated for up to 7 days and if the system still indicated negative, a Gram stain was performed and a final subculture was done before reporting the specimen as negative. Oxidase-negative, lactose non-fermenting colonies, were screened using API 10S at the start of the outbreak. Later an abbreviated panel of biochemical tests [10] was used. Isolates biochemically typical of S. Typhi were serotyped using slide agglutination with S. polyvalent O, S. polyvalent H, S. O factor 9 (group D), S. H factor d and S. Vi antisera. A set of 30 S. Typhi isolates were sent to the U.S. Centers for Disease Control and Prevention (CDC) for confirmation and antimicrobial susceptibility testing (AST). The National Antimicrobial Resistance Monitoring System at CDC performed AST on 17 isolates by broth microdilution to determine minimum inhibitory concentrations for 14 antimicrobial agents: amoxicillin/clavulanic acid, ampicillin, azithromycin, ceftiofur, ceftriaxone, cefoxitin, chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Results were interpreted using Clinical and Laboratory Standards Institute standards [11] when available. During the case-control study, we collected 5– 10 mL of blood from each of 20 suspected casepatients, placed the samples into commercial BD Bactecâ„¢ Aerobic/F media, and transported them to the clinical laboratory at the Makerere College of Health Sciences Department of Medical Microbiology for incubation in the Bactec 9120â„¢ blood culture system. Subcultures onto MacConkey and blood agar were done following instrument signals of growth or at the end of 7 days of incubation. Colonies were identified as S. Typhi based on biochemical characteristics including motility, hydrogen sulfide production, fermentation of sugars, urease production, and serological typing characteristics with various specific antisera. Environmental laboratory investigation From 2 to 8 April, juice samples were collected from the Nakasero, Owino, and Shauriyako markets, and 100 mL water samples were collected from unprotected water sources such as unprotected springs (i.e., underground water sources that do not have barriers protecting them from contamination and run-off) and commercial vendors in Kampala Central Division. We chose these water collection sites because we observed people in the outbreak-affected areas collecting water from these sites. The juice samples were tested because case-persons said they usually consumed these drinks. We collected nine juice samples, including 3 “bushera†(millet and yeast), 2 “munanansi†(pineapple juice with tea leaves), 3 “butunda†(passion fruit), and 1 “bongo†(unpasteurized yogurt drink). We also collected 13 water samples, including 3 “kaveera†(water packaged and sold in a small plastic bag), one unlabeled bottle of water from a street vendor, water from three storage tanks, and water from five unprotected springs. Juice and water samples were tested using a modified version of the United States Environmental Protection Agency’s Standard Analytical Protocol for S. Typhi in Drinking Water [12]. Briefly, 125 mL of specimen was preenriched in 125 mL of double strength buffered peptone water at 37 °C, followed by parallel enrichment in Selenite Cysteine broth at 37 °C and RV broth at 42 °C. Cultures from Selenite Cysteine broth were plated onto MacConkey and XLD agars; cultures from RV broth were plated onto XLD agar. All plates were incubated at 37 °C. Plates were inspected at 24 and 48 h for colony morphology consistent with Enterobacteriacea. Colonies morphologically consistent with S. spp. (i.e. lactose negative) were subjected to biochemical testing. Suspect isolates were sent to CDC-Atlanta for biochemical confirmation. For confirmation, suspect cultures were streaked onto Hektoen enteric agar and suspect colonies were subjected to an abbreviated panel of tests, for phenotypic identification of Salmonella or Shigella spp. and biochemical differentiation of S. serovars Typhi and Paratyphi A from other Salmonella serovars [10]. Statistical analysis Using surveillance data, the attack rates by sex, division, and sex were calculated using population data from the national census [8] and data provided by the Uganda Bureau of Statistics [13]. Using the StatCalc in Epi Info 7, considering a power of 80%, two sided confidence level of 95%, a case-control ratio of 1:2 with 30% of cases exposed and 10% of controls exposed, we would require about 39 cases and 77 controls. To measure the associations between exposure variables and illness status, we used the Mantel-Haenszel method to estimate odds ratios (OR) and their confidence intervals, accounting for frequency-matching of cases and controls. We calculated the proportion of cases and controls who drank 1, 2, and 3–4 types of locally made drinks, and used the Chisquare test for linear trend to assess the relationship between the number of types of drinks consumed and odds of illness [14]. Results Surveillance From 17 February to 12 June 2015, we identified 10,230 suspected cases from the six treatment centers established by MoH. Cases were distributed widely throughout Kampala and neighboring areas (Fig. 1). The epidemic curve of suspected cases suggests that the outbreak started at the beginning of February or perhaps earlier. By the time the outbreak was recognized on 6 February, hundreds of cases had already occurred (Fig. 2). Cases were reported in all five divisions of Kampala: Makindye (32%, 3234), Rubaga (28%, 2828), Kawempe (11%, 1144), Nakawa (6.4%, 656) and Central (4.2%, 428); for 19% (1940) of cases, either no division of residence was identified, or resided outside of Kampala. The attack rate during the outbreak period was highest in Makindye (10/1,000), Rubaga (8.7/1000), and Central (6.5/1000) Divisions. Males had a higher attack rate than females. The attack rate among people in the 15–59 year age group (12/1000) was 6 times higher than among younger (2.0/1000) or older (2.0/1000) persons (Table 1). Case-control study In our hypothesis-generating interviews of patients from the area where the outbreak was first identified, consumption of drinks made with water extracted from unprotected sources and packed in unhygienic conditions was often reported. Of the 33 case-patients we enrolled in the case-control study, 60% were men; the majority of the case-patients (85%) were in the 20–39 year and 9.1% in older age groups. In addition to fever, commonly reported symptoms included abdominal pain (72.72%) and headache (69.69%) (Table 2). We found that 22.58% (7/31) of case patients compared with 2.56% (2/78) of controls usually drank locally packaged water in small plastic bags called “kaveera water†(ORM-H = 8.90; 95% CI = 1.60–49.00); 55% (18/33) of case-patients compared with 19.23% (15/78) of controls drank locally-made passion fruit juice called “butunda†(ORM-H = 4.60; 95% CI: 1.90– 11.00); 31.25% (10/32) of case-patients compared with 16.67% (13/78) of controls usually drank locally-packed pineapple juice called “munanansi†(ORM-H = 2.00; 95% CI = 0.74–5.20); and 15.63% (5/32) of case-patients compared with 8.97% (7/78) of controls usually drank cold millet porridge called “bushera†(ORM-H = 2.80; 95% CI = 0.76–10.00). Workplace as a source of breakfast (ORM-H = 0.25; 95% CI = 0.07–0.93), and workplace as a source of lunch (ORM-H = 0.35; 95% CI = 0.11–1.10) were not significant risk factors for illness. When we compared the proportions of case-patients and controls who drank 0, 1, 2, or 3–4 types of locallymade drinks, we found that case-patients were more likely to drink multiple types of locally-made drinks than controls (Chi-square for linear trend = 14.65, p < 0.001) (Table 3). Laboratory investigation Of 10,230 suspected cases, 3464 (10%) underwent TUBEX® TF testing. Of those, 1038 were positive, representing a positivity rate of 29%. Blood samples from a total of 364 patients (including 20 of 33 case-control study patients) were tested by blood culture and 56 (15%) (including 5 of the 20 case-control study patients tested) yielded S. enterica ser. Typhi. Subsequently, 30 of the 56 S. Typhi isolates from blood cultures were confirmed at the U.S. CDC as S. Typhi. CDC determined the minimum inhibitory concentrations for 17 of these isolates, 5 of which were resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, nalidixic acid, trimethoprim/sulfamethoxazole and had intermediate interpretation to ciprofloxacin. The remaining 12 were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. Environmental investigation One of 3 “kaveera water†samples and the unlabeled bottle of water sold by street vendors contained lactose fermenting bacteria, which are commonly Enterobacteriaceae and associated with fecal contamination. The 2 other “kaveera water†samples contained non-lactose fermenting bacteria, also consistent with fecal contamination, and one isolate was further identified as non-typhoidal Salmonella. The 5 water samples from unprotected springs showed evidence of robust contamination with lactose fermenting bacteria. Lactose non-fermenting colonies from 2 spring water samples were identified as non-typhoidal Salmonella spp. Lactose fermenting bacteria were also detected in 2 of 3 “bushera†samples, 1 of 2 “munanasi†samples, all 3 “munanansi†juice samples, and the “bongo†sample. Additionally, non-typhoidal Salmonella was cultured from 1 “busheraâ€, 1 “munanansiâ€, and 1 passion fruit juice sample. Discussion Our investigation revealed a prolonged and widespread outbreak of typhoid fever that affected thousands of people in all five divisions of Kampala City over several months. Contaminated water from unprotected sources and drinks made with it were the likely vehicles of infection early in the outbreak. Juice and water samples obtained from street vendors and water samples collected from unprotected spring water sources showed evidence of fecal contamination. Although S. Typhi was not recovered from environmental testing, non-Typhi Salmonella were isolated from five street-vended beverage samples from the implicated markets. All 17 isolates of S. Typhi from blood tested at CDC were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. It is possible that persons affected by the antibiotic-resistant strains during this outbreak had experienced complications of typhoid fever considering that antibiotic-resistant strains of S. Typhi are associated with more severe form of the illness, complications and death [15]. This outbreak may have started in January 2015 or even earlier; however, it was not recognized until early February because routine clinical and laboratory surveillance systems for typhoid fever were not in place before the investigation. The widespread nature of the outbreak is compatible with a waterborne source. The sudden increase in cases after the start of the investigation was likely due to active community outreach and education about the symptoms of typhoid fever and the availability of prompt, free diagnostic testing and treatment through newly established treatment centers. The gradual decline in cases from mid-March onwards was likely the result of patient treatment and public health interventions including provision of free water chlorination products, sensitization of residents on water treatment, and the establishment of free alternative safe water sources in the most affected communities. Based on the evidence we presented, the Kampala Capital City Authority sealed off all underground water sources and worked with the National Water and Sewerage Corporation to ensure the provision of accessible alternative sources of water to the affected communities. In Uganda, as in many low and middle income countries, definitive diagnostic tests for typhoid fever such as blood culture are usually unavailable, unaffordable, or inconsistently applied [16]. Instead, typhoid fever diagnosis and surveillance often rely on clinical judgment or on the Widal test, which has poor sensitivity and specificity [17]. Moreover, physicians often give presumptive antibiotic and/or antimalarial treatment for febrile illnesses [18, 19] without attempting to determine the etiology. Previous studies have indicated that a significant proportion of febrile illness in Uganda is caused by bacteremia, including invasive non-Typhi salmonellosis and typhoid fever [20]. A more robust approach in these settings could entail periodically identifying persons with febrile illness in the communities and taking blood culture for confirmation [21]. The blood samples could be collected and sent using a specialized transport network to regional laboratory centers around the country where confirmative tests can be performed. This system has been successfully used to improve diagnostic services in early infant HIV/AIDS diagnosis [22]. Sentinel surveillance for febrile illnesses based on blood cultures would accelerate the early identification of outbreaks and implementation of control measures. Waterborne typhoid and paratyphoid fever affect an estimated 27 million people worldwide each year [3]. In developing countries, where safe water and sanitation systems have not been well-established, large-scale typhoid and paratyphoid outbreaks sometimes occur [7, 23–26]. During a previous typhoid outbreak in Kasese and Bundibugyo districts, Uganda, in 2009–2011, which affected 8092 persons, the vehicle of transmission was also found to be unclean water [7]. The current outbreak was likely caused by consuming contaminated water from unprotected ground water sources. Kampala city has more than 200 unprotected ground water sources, most of which serve as unprotected sources of water for economically disadvantaged people in the city such as those in our investigation [27]. Unsafe disposal of excreta and solid waste are significant factors that contribute to contamination of ground water in Kampala [28]. This outbreak investigation highlights the importance of ensuring access to affordable, safe, treated drinking water and improved sanitation and waste management systems for resource-constrained urban populations. Risk factors for typhoid transmission were not assessed later during this outbreak, when foodborne transmission might have become more common. Recurrent contamination of unprotected water sources with S. Typhi likely continued to sustain the outbreak propagation over the course of several months. According to the Uganda Demographic Health Survey 2011 [29], almost 30% of people living in urban areas and more than 60% of those living in rural areas do not treat their water before drinking it. Barriers to safer drinking water include the cost associated with establishing a piped treated water system or purchasing water treatment products for household use and the false perception that naturally occurring water sources could be safe [30]. In the aftermath of outbreaks like this one, public health authorities face 3 possible options: The first option is to do nothing but respond to outbreaks as they occur. Governments in resource-constrained settings often choose this option, leaving the population vulnerable to outbreaks of waterborne diseases including cholera, hepatitis A and E, cryptosporidiosis, shigellosis, and many others in addition to typhoid and paratyphoid fever. The second option is mass vaccination against typhoid fever. A cost-effectiveness evaluation of a hypothetical typhoid vaccination campaign was carried out after the multi-year outbreak of typhoid fever in Kasese District, Uganda, and it was estimated to be highly costeffective [31]. However, vaccination against the many different pathogens that cause waterborne diseases is not possible because vaccines are not yet available for many of them (e.g. cryptosporidiosis, shigellosis, paratyphoid fever, etc.). In addition, typhoid fever vaccines have been shown to have varied levels of effectiveness (from 50 to 95%) and to last for varied lengths of time (from 3 to 10 years) [15]. The third and final option is to improve the water and sanitation systems. Improvement of sanitation, hygiene and clean water supply around the world could avert ≥90% of diarrheal disease episodes annually [32]. In North America and Europe, typhoid fever caused largescale outbreaks from the late 19th through the early 20th century [33, 34]. After improvement of municipal water and sanitation systems in the early 20th century, waterborne outbreaks of communicable diseases including typhoid fever drastically decreased [35–37]. Improved sanitation measures such as having a basic pit latrine or a toilet connected to a septic tank curtail the direct contact between human waste and water or the environment. Yet in 2012, only 33% of the urban population in Uganda had access to adequate sanitation, an increase of only 1% since 1990, and 2% still practiced open defecation [38]. Although improving water and sanitation systems requires a substantial investment by the government, ultimately it is highly cost-effective in the reduction of many waterborne diseases [39]. Strengths and limitations A major limitation of our investigation was that, due to inadequate laboratory capacity to confirm a large number of cases early in the outbreak, and the need to rapidly identify the mode of transmission to inform effective interventions, we included non-laboratory confirmed cases in our case-control study. While a clinical case definition for typhoid fever cases can lead to misclassification, and is not recommended during nonoutbreak situations, during an outbreak such a case definition will often perform well, as measured by good positive and negative predictive values [40]. Also, the information on usual sources of water and food was based on self-reports, which could represent a source ofinformation bias. Another limitation is that data on mortality or on complications such as intestinal perforations were not collected. With over 10,000 cases it is likely that there were intestinal perforations and deaths but no surveillance for those outcomes was done. A study is currently being conducted to assess these severe impacts of this outbreak. In addition, only a few water and juice samples were tested, which could explain why S. Typhi was not isolated during the environmental investigation. Also, water and juice samples were tested using the reagents and procedures that were available in the laboratory for testing clinical specimens for Salmonella, and not more conventional methods for evaluating the potential presence of fecal contamination in these types of samples. Conclusion In conclusion, this investigation revealed a large outbreak of typhoid fever that affected thousands of people in Kampala, Uganda, which appeared to have been caused by consuming contaminated water and local drinks made from it. To prevent future waterborne outbreaks, we recommended that the Kampala Capital City Authority, the MoH, the National Water and Sewerage Corporation, and partners invest in improving access to potable water, and safe sanitation and hygiene facilities [41].

When did this event start?

198

A large and persistent outbreak of typhoid fever caused by consuming contaminated water and street-vended beverages Kampala, Uganda, January

Abstract Background: On 6 February 2015, Kampala city authorities alerted the Ugandan Ministry of Health of a “strange disease†that killed one person and sickened dozens. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods: We defined a suspected case as onset of fever (≥37.5 °C) for more than 3 days with abdominal pain, headache, negative malaria test or failed anti-malaria treatment, and at least 2 of the following: diarrhea, nausea or vomiting, constipation, fatigue. A probable case was defined as a suspected case with a positive TUBEX® TF test. A confirmed case had blood culture yielding Salmonella Typhi. We conducted a case-control study to compare exposures of 33 suspected case-patients and 78 controls, and tested water and juice samples. Results: From 17 February-12 June, we identified 10,230 suspected, 1038 probable, and 51 confirmed cases. Approximately 22.58% (7/31) of case-patients and 2.56% (2/78) of controls drank water sold in small plastic bags (ORM-H = 8.90; 95%CI = 1.60–49.00); 54.54% (18/33) of case-patients and 19.23% (15/78) of controls consumed locallymade drinks (ORM-H = 4.60; 95%CI: 1.90–11.00). All isolates were susceptible to ciprofloxacin and ceftriaxone. Water and juice samples exhibited evidence of fecal contamination. Conclusion: Contaminated water and street-vended beverages were likely vehicles of this outbreak. At our recommendation authorities closed unsafe water sources and supplied safe water to affected areas. Keywords: Typhoid fever, Outbreak, Case-control, Uganda Background Typhoid fever is a systemic disease caused by Salmonella enterica serovar Typhi, a Gram-negative bacterium. Humans are the only host, and transmission most commonly occurs through ingestion of water or food contaminated by feces from an acutely ill or convalescent patient or an asymptomatic carrier. The incubation period is usually 1 to 2 weeks but can range from 3 to 60 days [1]. The illness presents with sustained fever and a constellation of other symptoms including dry cough, fatigue, abdominal pain, diarrhea, and constipation [2]. Case fatality ratios range between 10 and 30% if untreated, but fall to 1–4% with appropriate and timely antimicrobial treatment [3]. The gold standard laboratory diagnosis of typhoid fever requires isolation of S.. Typhi from blood, stool, bone marrow, or other tissue or bodily fluid by bacterial culture [2]. Other tests with moderate sensitivity and specificity include the Widal test and TUBEX® TF test which involve detection of antibodies against S. Typhi antigens [2]. Typhoid fever is preventable through public health interventions such as provision of safe water, ensuring proper sanitation and waste disposal systems, and excluding disease carriers from handling food [4]. Typhoid fever is a major cause of mortality and morbidity worldwide. In endemic areas, the disease is most commonly found in children 5–19 years of age. International visitors from non-endemic areas are also at risk if unvaccinated [1]. The global burden of the disease in low- and middle-income countries in 2010 was estimated to be 11.9 million cases, including 129,000 fatalities, after adjusting for water-related risk factors [5]. In Uganda, an outbreak of typhoid fever in Kasese District sickened 8092 persons from 27 December 2007 to 30 July 2009, resulting in at least 249 intestinal perforations and 47 deaths [6]. In 2011, numerous typhoid cases were again reported in Kasese and neighboring Bundibugyo District with many more intestinal perforations and emergence of multidrug resistant strains [7]. On 6 February, 2015, the Ugandan Ministry of Health (MoH) received a report from the Kampala Capital City Authority that a 42-year-old man had died a day earlier of a “strange illness.†The patient was admitted to the hospital on 2 February 2015 with symptoms of abdominal pain, high fever, and severe jaundice. Initial testing involved use of the Widal test which was positive. Approximately 30 other people who worked in the same area as the deceased reportedly had similar symptoms. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods Study sites The outbreak occurred in Kampala (estimated population 1.4 million), the capital of Uganda [8]. Kampala has five divisions: Kampala Central, Kawempe, Makindye, Rubaga, and Nakawa. We focused our epidemiologic investigation on two markets and a commuter taxi park in Kampala Central Division where the initial cases were concentrated. Surveillance To characterize and control the epidemic, MoH conducted surveillance at six treatment centers established in affected areas of the city to provide diagnostic testing and typhoid fever treatment free of charge. These treatment centers were existing health centers in which routine disease surveillance and treatment activities are conducted. Through the media, local leaders encouraged the people with symptoms of typhoid fever to seek medical care at these treatment centers. We defined a suspected case as onset of fever (≥37.5 °C) for ≥3 days from 1 January 2015 onwards, with headache, abdominal pain, a negative test for malaria or failure to respond to anti-malaria treatment, and ≥2 of the following symptoms: diarrhea, nausea or vomiting, constipation, or fatigue. A probable case was a suspected case whose serum sample yielded a positive TUBEX® TF test [9]. Blood samples were collected from the first 5 suspected cases every day from each treatment center and referred to the microbiology laboratory at the Medical Research Council for blood culture. A confirmed case was a suspected case whose blood culture yielded S. Typhi. Case-control study We conducted open-ended hypothesis-generating interviews of case-patients found in the areas where the initial cases were identified, focusing on their usual sources of water and food. To test the hypotheses generated from the interviews, we conducted a case-control study from 10 to 20 February 2015. To rapidly identify the mode of transmission so as to inform prompt prevention and control measures, we used the initial 33 suspected case-patients identified in the earliest-affected communities for the case-control study. The earliest cases were persons working in two markets or in the central terminal station for Kampala’s shared taxis, all of which were located in central Kampala. Therefore we recruited both the cases and the controls from those places. The markets are open spaces where people set up their stalls to sell assorted merchandise, whereas the central terminal station for the shared taxis is an area where the shared taxis (mini-vans) pick up and drop off passengers. In the markets, after identifying and interviewing a case, the interviewer then walked around the stall to identify several persons of the same gender and similar age as the case from the surrounding stalls who never had a febrile disease since January 1, 2015, and recruited those persons as controls. Similarly, in the central terminal station for the shared taxis, after identifying and interviewing a case who was working inside a shared taxi (e.g., a driver or conductor), the interviewer then walked around the shared taxi to recruit asymptomatic workers of the same gender and similar age from the surrounding shared taxis as controls. The interviewers used a structured questionnaire to collect information on the usual water and food exposures from the case- and control-persons. A link to the questionnaire that was used has been provided in the Additional file 1 of the manuscript. Clinical laboratory investigation The TUBEX® TF test was performed at the treatment centers by trained clinical and laboratory staff as per the manufacturer’s instructions. Blood culture was performed on the first five patients presenting each day at the 6 treatment centers. From each adult patient, 5–10 mL of blood was collected and inoculated in BD Bactecâ„¢ Aerobic/F blood culture bottles and incubated in a BD Bactec 9000 seriesâ„¢. Presumptive positive bottles, as signaled by the system, were subcultured on MacConkey, chocolate, and blood agar plates and incubated aerobically at 37 °C for 24 h. A Gram stain was also performed. Negative vials were incubated for up to 7 days and if the system still indicated negative, a Gram stain was performed and a final subculture was done before reporting the specimen as negative. Oxidase-negative, lactose non-fermenting colonies, were screened using API 10S at the start of the outbreak. Later an abbreviated panel of biochemical tests [10] was used. Isolates biochemically typical of S. Typhi were serotyped using slide agglutination with S. polyvalent O, S. polyvalent H, S. O factor 9 (group D), S. H factor d and S. Vi antisera. A set of 30 S. Typhi isolates were sent to the U.S. Centers for Disease Control and Prevention (CDC) for confirmation and antimicrobial susceptibility testing (AST). The National Antimicrobial Resistance Monitoring System at CDC performed AST on 17 isolates by broth microdilution to determine minimum inhibitory concentrations for 14 antimicrobial agents: amoxicillin/clavulanic acid, ampicillin, azithromycin, ceftiofur, ceftriaxone, cefoxitin, chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Results were interpreted using Clinical and Laboratory Standards Institute standards [11] when available. During the case-control study, we collected 5– 10 mL of blood from each of 20 suspected casepatients, placed the samples into commercial BD Bactecâ„¢ Aerobic/F media, and transported them to the clinical laboratory at the Makerere College of Health Sciences Department of Medical Microbiology for incubation in the Bactec 9120â„¢ blood culture system. Subcultures onto MacConkey and blood agar were done following instrument signals of growth or at the end of 7 days of incubation. Colonies were identified as S. Typhi based on biochemical characteristics including motility, hydrogen sulfide production, fermentation of sugars, urease production, and serological typing characteristics with various specific antisera. Environmental laboratory investigation From 2 to 8 April, juice samples were collected from the Nakasero, Owino, and Shauriyako markets, and 100 mL water samples were collected from unprotected water sources such as unprotected springs (i.e., underground water sources that do not have barriers protecting them from contamination and run-off) and commercial vendors in Kampala Central Division. We chose these water collection sites because we observed people in the outbreak-affected areas collecting water from these sites. The juice samples were tested because case-persons said they usually consumed these drinks. We collected nine juice samples, including 3 “bushera†(millet and yeast), 2 “munanansi†(pineapple juice with tea leaves), 3 “butunda†(passion fruit), and 1 “bongo†(unpasteurized yogurt drink). We also collected 13 water samples, including 3 “kaveera†(water packaged and sold in a small plastic bag), one unlabeled bottle of water from a street vendor, water from three storage tanks, and water from five unprotected springs. Juice and water samples were tested using a modified version of the United States Environmental Protection Agency’s Standard Analytical Protocol for S. Typhi in Drinking Water [12]. Briefly, 125 mL of specimen was preenriched in 125 mL of double strength buffered peptone water at 37 °C, followed by parallel enrichment in Selenite Cysteine broth at 37 °C and RV broth at 42 °C. Cultures from Selenite Cysteine broth were plated onto MacConkey and XLD agars; cultures from RV broth were plated onto XLD agar. All plates were incubated at 37 °C. Plates were inspected at 24 and 48 h for colony morphology consistent with Enterobacteriacea. Colonies morphologically consistent with S. spp. (i.e. lactose negative) were subjected to biochemical testing. Suspect isolates were sent to CDC-Atlanta for biochemical confirmation. For confirmation, suspect cultures were streaked onto Hektoen enteric agar and suspect colonies were subjected to an abbreviated panel of tests, for phenotypic identification of Salmonella or Shigella spp. and biochemical differentiation of S. serovars Typhi and Paratyphi A from other Salmonella serovars [10]. Statistical analysis Using surveillance data, the attack rates by sex, division, and sex were calculated using population data from the national census [8] and data provided by the Uganda Bureau of Statistics [13]. Using the StatCalc in Epi Info 7, considering a power of 80%, two sided confidence level of 95%, a case-control ratio of 1:2 with 30% of cases exposed and 10% of controls exposed, we would require about 39 cases and 77 controls. To measure the associations between exposure variables and illness status, we used the Mantel-Haenszel method to estimate odds ratios (OR) and their confidence intervals, accounting for frequency-matching of cases and controls. We calculated the proportion of cases and controls who drank 1, 2, and 3–4 types of locally made drinks, and used the Chisquare test for linear trend to assess the relationship between the number of types of drinks consumed and odds of illness [14]. Results Surveillance From 17 February to 12 June 2015, we identified 10,230 suspected cases from the six treatment centers established by MoH. Cases were distributed widely throughout Kampala and neighboring areas (Fig. 1). The epidemic curve of suspected cases suggests that the outbreak started at the beginning of February or perhaps earlier. By the time the outbreak was recognized on 6 February, hundreds of cases had already occurred (Fig. 2). Cases were reported in all five divisions of Kampala: Makindye (32%, 3234), Rubaga (28%, 2828), Kawempe (11%, 1144), Nakawa (6.4%, 656) and Central (4.2%, 428); for 19% (1940) of cases, either no division of residence was identified, or resided outside of Kampala. The attack rate during the outbreak period was highest in Makindye (10/1,000), Rubaga (8.7/1000), and Central (6.5/1000) Divisions. Males had a higher attack rate than females. The attack rate among people in the 15–59 year age group (12/1000) was 6 times higher than among younger (2.0/1000) or older (2.0/1000) persons (Table 1). Case-control study In our hypothesis-generating interviews of patients from the area where the outbreak was first identified, consumption of drinks made with water extracted from unprotected sources and packed in unhygienic conditions was often reported. Of the 33 case-patients we enrolled in the case-control study, 60% were men; the majority of the case-patients (85%) were in the 20–39 year and 9.1% in older age groups. In addition to fever, commonly reported symptoms included abdominal pain (72.72%) and headache (69.69%) (Table 2). We found that 22.58% (7/31) of case patients compared with 2.56% (2/78) of controls usually drank locally packaged water in small plastic bags called “kaveera water†(ORM-H = 8.90; 95% CI = 1.60–49.00); 55% (18/33) of case-patients compared with 19.23% (15/78) of controls drank locally-made passion fruit juice called “butunda†(ORM-H = 4.60; 95% CI: 1.90– 11.00); 31.25% (10/32) of case-patients compared with 16.67% (13/78) of controls usually drank locally-packed pineapple juice called “munanansi†(ORM-H = 2.00; 95% CI = 0.74–5.20); and 15.63% (5/32) of case-patients compared with 8.97% (7/78) of controls usually drank cold millet porridge called “bushera†(ORM-H = 2.80; 95% CI = 0.76–10.00). Workplace as a source of breakfast (ORM-H = 0.25; 95% CI = 0.07–0.93), and workplace as a source of lunch (ORM-H = 0.35; 95% CI = 0.11–1.10) were not significant risk factors for illness. When we compared the proportions of case-patients and controls who drank 0, 1, 2, or 3–4 types of locallymade drinks, we found that case-patients were more likely to drink multiple types of locally-made drinks than controls (Chi-square for linear trend = 14.65, p < 0.001) (Table 3). Laboratory investigation Of 10,230 suspected cases, 3464 (10%) underwent TUBEX® TF testing. Of those, 1038 were positive, representing a positivity rate of 29%. Blood samples from a total of 364 patients (including 20 of 33 case-control study patients) were tested by blood culture and 56 (15%) (including 5 of the 20 case-control study patients tested) yielded S. enterica ser. Typhi. Subsequently, 30 of the 56 S. Typhi isolates from blood cultures were confirmed at the U.S. CDC as S. Typhi. CDC determined the minimum inhibitory concentrations for 17 of these isolates, 5 of which were resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, nalidixic acid, trimethoprim/sulfamethoxazole and had intermediate interpretation to ciprofloxacin. The remaining 12 were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. Environmental investigation One of 3 “kaveera water†samples and the unlabeled bottle of water sold by street vendors contained lactose fermenting bacteria, which are commonly Enterobacteriaceae and associated with fecal contamination. The 2 other “kaveera water†samples contained non-lactose fermenting bacteria, also consistent with fecal contamination, and one isolate was further identified as non-typhoidal Salmonella. The 5 water samples from unprotected springs showed evidence of robust contamination with lactose fermenting bacteria. Lactose non-fermenting colonies from 2 spring water samples were identified as non-typhoidal Salmonella spp. Lactose fermenting bacteria were also detected in 2 of 3 “bushera†samples, 1 of 2 “munanasi†samples, all 3 “munanansi†juice samples, and the “bongo†sample. Additionally, non-typhoidal Salmonella was cultured from 1 “busheraâ€, 1 “munanansiâ€, and 1 passion fruit juice sample. Discussion Our investigation revealed a prolonged and widespread outbreak of typhoid fever that affected thousands of people in all five divisions of Kampala City over several months. Contaminated water from unprotected sources and drinks made with it were the likely vehicles of infection early in the outbreak. Juice and water samples obtained from street vendors and water samples collected from unprotected spring water sources showed evidence of fecal contamination. Although S. Typhi was not recovered from environmental testing, non-Typhi Salmonella were isolated from five street-vended beverage samples from the implicated markets. All 17 isolates of S. Typhi from blood tested at CDC were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. It is possible that persons affected by the antibiotic-resistant strains during this outbreak had experienced complications of typhoid fever considering that antibiotic-resistant strains of S. Typhi are associated with more severe form of the illness, complications and death [15]. This outbreak may have started in January 2015 or even earlier; however, it was not recognized until early February because routine clinical and laboratory surveillance systems for typhoid fever were not in place before the investigation. The widespread nature of the outbreak is compatible with a waterborne source. The sudden increase in cases after the start of the investigation was likely due to active community outreach and education about the symptoms of typhoid fever and the availability of prompt, free diagnostic testing and treatment through newly established treatment centers. The gradual decline in cases from mid-March onwards was likely the result of patient treatment and public health interventions including provision of free water chlorination products, sensitization of residents on water treatment, and the establishment of free alternative safe water sources in the most affected communities. Based on the evidence we presented, the Kampala Capital City Authority sealed off all underground water sources and worked with the National Water and Sewerage Corporation to ensure the provision of accessible alternative sources of water to the affected communities. In Uganda, as in many low and middle income countries, definitive diagnostic tests for typhoid fever such as blood culture are usually unavailable, unaffordable, or inconsistently applied [16]. Instead, typhoid fever diagnosis and surveillance often rely on clinical judgment or on the Widal test, which has poor sensitivity and specificity [17]. Moreover, physicians often give presumptive antibiotic and/or antimalarial treatment for febrile illnesses [18, 19] without attempting to determine the etiology. Previous studies have indicated that a significant proportion of febrile illness in Uganda is caused by bacteremia, including invasive non-Typhi salmonellosis and typhoid fever [20]. A more robust approach in these settings could entail periodically identifying persons with febrile illness in the communities and taking blood culture for confirmation [21]. The blood samples could be collected and sent using a specialized transport network to regional laboratory centers around the country where confirmative tests can be performed. This system has been successfully used to improve diagnostic services in early infant HIV/AIDS diagnosis [22]. Sentinel surveillance for febrile illnesses based on blood cultures would accelerate the early identification of outbreaks and implementation of control measures. Waterborne typhoid and paratyphoid fever affect an estimated 27 million people worldwide each year [3]. In developing countries, where safe water and sanitation systems have not been well-established, large-scale typhoid and paratyphoid outbreaks sometimes occur [7, 23–26]. During a previous typhoid outbreak in Kasese and Bundibugyo districts, Uganda, in 2009–2011, which affected 8092 persons, the vehicle of transmission was also found to be unclean water [7]. The current outbreak was likely caused by consuming contaminated water from unprotected ground water sources. Kampala city has more than 200 unprotected ground water sources, most of which serve as unprotected sources of water for economically disadvantaged people in the city such as those in our investigation [27]. Unsafe disposal of excreta and solid waste are significant factors that contribute to contamination of ground water in Kampala [28]. This outbreak investigation highlights the importance of ensuring access to affordable, safe, treated drinking water and improved sanitation and waste management systems for resource-constrained urban populations. Risk factors for typhoid transmission were not assessed later during this outbreak, when foodborne transmission might have become more common. Recurrent contamination of unprotected water sources with S. Typhi likely continued to sustain the outbreak propagation over the course of several months. According to the Uganda Demographic Health Survey 2011 [29], almost 30% of people living in urban areas and more than 60% of those living in rural areas do not treat their water before drinking it. Barriers to safer drinking water include the cost associated with establishing a piped treated water system or purchasing water treatment products for household use and the false perception that naturally occurring water sources could be safe [30]. In the aftermath of outbreaks like this one, public health authorities face 3 possible options: The first option is to do nothing but respond to outbreaks as they occur. Governments in resource-constrained settings often choose this option, leaving the population vulnerable to outbreaks of waterborne diseases including cholera, hepatitis A and E, cryptosporidiosis, shigellosis, and many others in addition to typhoid and paratyphoid fever. The second option is mass vaccination against typhoid fever. A cost-effectiveness evaluation of a hypothetical typhoid vaccination campaign was carried out after the multi-year outbreak of typhoid fever in Kasese District, Uganda, and it was estimated to be highly costeffective [31]. However, vaccination against the many different pathogens that cause waterborne diseases is not possible because vaccines are not yet available for many of them (e.g. cryptosporidiosis, shigellosis, paratyphoid fever, etc.). In addition, typhoid fever vaccines have been shown to have varied levels of effectiveness (from 50 to 95%) and to last for varied lengths of time (from 3 to 10 years) [15]. The third and final option is to improve the water and sanitation systems. Improvement of sanitation, hygiene and clean water supply around the world could avert ≥90% of diarrheal disease episodes annually [32]. In North America and Europe, typhoid fever caused largescale outbreaks from the late 19th through the early 20th century [33, 34]. After improvement of municipal water and sanitation systems in the early 20th century, waterborne outbreaks of communicable diseases including typhoid fever drastically decreased [35–37]. Improved sanitation measures such as having a basic pit latrine or a toilet connected to a septic tank curtail the direct contact between human waste and water or the environment. Yet in 2012, only 33% of the urban population in Uganda had access to adequate sanitation, an increase of only 1% since 1990, and 2% still practiced open defecation [38]. Although improving water and sanitation systems requires a substantial investment by the government, ultimately it is highly cost-effective in the reduction of many waterborne diseases [39]. Strengths and limitations A major limitation of our investigation was that, due to inadequate laboratory capacity to confirm a large number of cases early in the outbreak, and the need to rapidly identify the mode of transmission to inform effective interventions, we included non-laboratory confirmed cases in our case-control study. While a clinical case definition for typhoid fever cases can lead to misclassification, and is not recommended during nonoutbreak situations, during an outbreak such a case definition will often perform well, as measured by good positive and negative predictive values [40]. Also, the information on usual sources of water and food was based on self-reports, which could represent a source ofinformation bias. Another limitation is that data on mortality or on complications such as intestinal perforations were not collected. With over 10,000 cases it is likely that there were intestinal perforations and deaths but no surveillance for those outcomes was done. A study is currently being conducted to assess these severe impacts of this outbreak. In addition, only a few water and juice samples were tested, which could explain why S. Typhi was not isolated during the environmental investigation. Also, water and juice samples were tested using the reagents and procedures that were available in the laboratory for testing clinical specimens for Salmonella, and not more conventional methods for evaluating the potential presence of fecal contamination in these types of samples. Conclusion In conclusion, this investigation revealed a large outbreak of typhoid fever that affected thousands of people in Kampala, Uganda, which appeared to have been caused by consuming contaminated water and local drinks made from it. To prevent future waterborne outbreaks, we recommended that the Kampala Capital City Authority, the MoH, the National Water and Sewerage Corporation, and partners invest in improving access to potable water, and safe sanitation and hygiene facilities [41].

What is the date of this event?

199

A large and persistent outbreak of typhoid fever caused by consuming contaminated water and street-vended beverages Kampala, Uganda, January

Abstract Background: On 6 February 2015, Kampala city authorities alerted the Ugandan Ministry of Health of a “strange disease†that killed one person and sickened dozens. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods: We defined a suspected case as onset of fever (≥37.5 °C) for more than 3 days with abdominal pain, headache, negative malaria test or failed anti-malaria treatment, and at least 2 of the following: diarrhea, nausea or vomiting, constipation, fatigue. A probable case was defined as a suspected case with a positive TUBEX® TF test. A confirmed case had blood culture yielding Salmonella Typhi. We conducted a case-control study to compare exposures of 33 suspected case-patients and 78 controls, and tested water and juice samples. Results: From 17 February-12 June, we identified 10,230 suspected, 1038 probable, and 51 confirmed cases. Approximately 22.58% (7/31) of case-patients and 2.56% (2/78) of controls drank water sold in small plastic bags (ORM-H = 8.90; 95%CI = 1.60–49.00); 54.54% (18/33) of case-patients and 19.23% (15/78) of controls consumed locallymade drinks (ORM-H = 4.60; 95%CI: 1.90–11.00). All isolates were susceptible to ciprofloxacin and ceftriaxone. Water and juice samples exhibited evidence of fecal contamination. Conclusion: Contaminated water and street-vended beverages were likely vehicles of this outbreak. At our recommendation authorities closed unsafe water sources and supplied safe water to affected areas. Keywords: Typhoid fever, Outbreak, Case-control, Uganda Background Typhoid fever is a systemic disease caused by Salmonella enterica serovar Typhi, a Gram-negative bacterium. Humans are the only host, and transmission most commonly occurs through ingestion of water or food contaminated by feces from an acutely ill or convalescent patient or an asymptomatic carrier. The incubation period is usually 1 to 2 weeks but can range from 3 to 60 days [1]. The illness presents with sustained fever and a constellation of other symptoms including dry cough, fatigue, abdominal pain, diarrhea, and constipation [2]. Case fatality ratios range between 10 and 30% if untreated, but fall to 1–4% with appropriate and timely antimicrobial treatment [3]. The gold standard laboratory diagnosis of typhoid fever requires isolation of S.. Typhi from blood, stool, bone marrow, or other tissue or bodily fluid by bacterial culture [2]. Other tests with moderate sensitivity and specificity include the Widal test and TUBEX® TF test which involve detection of antibodies against S. Typhi antigens [2]. Typhoid fever is preventable through public health interventions such as provision of safe water, ensuring proper sanitation and waste disposal systems, and excluding disease carriers from handling food [4]. Typhoid fever is a major cause of mortality and morbidity worldwide. In endemic areas, the disease is most commonly found in children 5–19 years of age. International visitors from non-endemic areas are also at risk if unvaccinated [1]. The global burden of the disease in low- and middle-income countries in 2010 was estimated to be 11.9 million cases, including 129,000 fatalities, after adjusting for water-related risk factors [5]. In Uganda, an outbreak of typhoid fever in Kasese District sickened 8092 persons from 27 December 2007 to 30 July 2009, resulting in at least 249 intestinal perforations and 47 deaths [6]. In 2011, numerous typhoid cases were again reported in Kasese and neighboring Bundibugyo District with many more intestinal perforations and emergence of multidrug resistant strains [7]. On 6 February, 2015, the Ugandan Ministry of Health (MoH) received a report from the Kampala Capital City Authority that a 42-year-old man had died a day earlier of a “strange illness.†The patient was admitted to the hospital on 2 February 2015 with symptoms of abdominal pain, high fever, and severe jaundice. Initial testing involved use of the Widal test which was positive. Approximately 30 other people who worked in the same area as the deceased reportedly had similar symptoms. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. Methods Study sites The outbreak occurred in Kampala (estimated population 1.4 million), the capital of Uganda [8]. Kampala has five divisions: Kampala Central, Kawempe, Makindye, Rubaga, and Nakawa. We focused our epidemiologic investigation on two markets and a commuter taxi park in Kampala Central Division where the initial cases were concentrated. Surveillance To characterize and control the epidemic, MoH conducted surveillance at six treatment centers established in affected areas of the city to provide diagnostic testing and typhoid fever treatment free of charge. These treatment centers were existing health centers in which routine disease surveillance and treatment activities are conducted. Through the media, local leaders encouraged the people with symptoms of typhoid fever to seek medical care at these treatment centers. We defined a suspected case as onset of fever (≥37.5 °C) for ≥3 days from 1 January 2015 onwards, with headache, abdominal pain, a negative test for malaria or failure to respond to anti-malaria treatment, and ≥2 of the following symptoms: diarrhea, nausea or vomiting, constipation, or fatigue. A probable case was a suspected case whose serum sample yielded a positive TUBEX® TF test [9]. Blood samples were collected from the first 5 suspected cases every day from each treatment center and referred to the microbiology laboratory at the Medical Research Council for blood culture. A confirmed case was a suspected case whose blood culture yielded S. Typhi. Case-control study We conducted open-ended hypothesis-generating interviews of case-patients found in the areas where the initial cases were identified, focusing on their usual sources of water and food. To test the hypotheses generated from the interviews, we conducted a case-control study from 10 to 20 February 2015. To rapidly identify the mode of transmission so as to inform prompt prevention and control measures, we used the initial 33 suspected case-patients identified in the earliest-affected communities for the case-control study. The earliest cases were persons working in two markets or in the central terminal station for Kampala’s shared taxis, all of which were located in central Kampala. Therefore we recruited both the cases and the controls from those places. The markets are open spaces where people set up their stalls to sell assorted merchandise, whereas the central terminal station for the shared taxis is an area where the shared taxis (mini-vans) pick up and drop off passengers. In the markets, after identifying and interviewing a case, the interviewer then walked around the stall to identify several persons of the same gender and similar age as the case from the surrounding stalls who never had a febrile disease since January 1, 2015, and recruited those persons as controls. Similarly, in the central terminal station for the shared taxis, after identifying and interviewing a case who was working inside a shared taxi (e.g., a driver or conductor), the interviewer then walked around the shared taxi to recruit asymptomatic workers of the same gender and similar age from the surrounding shared taxis as controls. The interviewers used a structured questionnaire to collect information on the usual water and food exposures from the case- and control-persons. A link to the questionnaire that was used has been provided in the Additional file 1 of the manuscript. Clinical laboratory investigation The TUBEX® TF test was performed at the treatment centers by trained clinical and laboratory staff as per the manufacturer’s instructions. Blood culture was performed on the first five patients presenting each day at the 6 treatment centers. From each adult patient, 5–10 mL of blood was collected and inoculated in BD Bactecâ„¢ Aerobic/F blood culture bottles and incubated in a BD Bactec 9000 seriesâ„¢. Presumptive positive bottles, as signaled by the system, were subcultured on MacConkey, chocolate, and blood agar plates and incubated aerobically at 37 °C for 24 h. A Gram stain was also performed. Negative vials were incubated for up to 7 days and if the system still indicated negative, a Gram stain was performed and a final subculture was done before reporting the specimen as negative. Oxidase-negative, lactose non-fermenting colonies, were screened using API 10S at the start of the outbreak. Later an abbreviated panel of biochemical tests [10] was used. Isolates biochemically typical of S. Typhi were serotyped using slide agglutination with S. polyvalent O, S. polyvalent H, S. O factor 9 (group D), S. H factor d and S. Vi antisera. A set of 30 S. Typhi isolates were sent to the U.S. Centers for Disease Control and Prevention (CDC) for confirmation and antimicrobial susceptibility testing (AST). The National Antimicrobial Resistance Monitoring System at CDC performed AST on 17 isolates by broth microdilution to determine minimum inhibitory concentrations for 14 antimicrobial agents: amoxicillin/clavulanic acid, ampicillin, azithromycin, ceftiofur, ceftriaxone, cefoxitin, chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Results were interpreted using Clinical and Laboratory Standards Institute standards [11] when available. During the case-control study, we collected 5– 10 mL of blood from each of 20 suspected casepatients, placed the samples into commercial BD Bactecâ„¢ Aerobic/F media, and transported them to the clinical laboratory at the Makerere College of Health Sciences Department of Medical Microbiology for incubation in the Bactec 9120â„¢ blood culture system. Subcultures onto MacConkey and blood agar were done following instrument signals of growth or at the end of 7 days of incubation. Colonies were identified as S. Typhi based on biochemical characteristics including motility, hydrogen sulfide production, fermentation of sugars, urease production, and serological typing characteristics with various specific antisera. Environmental laboratory investigation From 2 to 8 April, juice samples were collected from the Nakasero, Owino, and Shauriyako markets, and 100 mL water samples were collected from unprotected water sources such as unprotected springs (i.e., underground water sources that do not have barriers protecting them from contamination and run-off) and commercial vendors in Kampala Central Division. We chose these water collection sites because we observed people in the outbreak-affected areas collecting water from these sites. The juice samples were tested because case-persons said they usually consumed these drinks. We collected nine juice samples, including 3 “bushera†(millet and yeast), 2 “munanansi†(pineapple juice with tea leaves), 3 “butunda†(passion fruit), and 1 “bongo†(unpasteurized yogurt drink). We also collected 13 water samples, including 3 “kaveera†(water packaged and sold in a small plastic bag), one unlabeled bottle of water from a street vendor, water from three storage tanks, and water from five unprotected springs. Juice and water samples were tested using a modified version of the United States Environmental Protection Agency’s Standard Analytical Protocol for S. Typhi in Drinking Water [12]. Briefly, 125 mL of specimen was preenriched in 125 mL of double strength buffered peptone water at 37 °C, followed by parallel enrichment in Selenite Cysteine broth at 37 °C and RV broth at 42 °C. Cultures from Selenite Cysteine broth were plated onto MacConkey and XLD agars; cultures from RV broth were plated onto XLD agar. All plates were incubated at 37 °C. Plates were inspected at 24 and 48 h for colony morphology consistent with Enterobacteriacea. Colonies morphologically consistent with S. spp. (i.e. lactose negative) were subjected to biochemical testing. Suspect isolates were sent to CDC-Atlanta for biochemical confirmation. For confirmation, suspect cultures were streaked onto Hektoen enteric agar and suspect colonies were subjected to an abbreviated panel of tests, for phenotypic identification of Salmonella or Shigella spp. and biochemical differentiation of S. serovars Typhi and Paratyphi A from other Salmonella serovars [10]. Statistical analysis Using surveillance data, the attack rates by sex, division, and sex were calculated using population data from the national census [8] and data provided by the Uganda Bureau of Statistics [13]. Using the StatCalc in Epi Info 7, considering a power of 80%, two sided confidence level of 95%, a case-control ratio of 1:2 with 30% of cases exposed and 10% of controls exposed, we would require about 39 cases and 77 controls. To measure the associations between exposure variables and illness status, we used the Mantel-Haenszel method to estimate odds ratios (OR) and their confidence intervals, accounting for frequency-matching of cases and controls. We calculated the proportion of cases and controls who drank 1, 2, and 3–4 types of locally made drinks, and used the Chisquare test for linear trend to assess the relationship between the number of types of drinks consumed and odds of illness [14]. Results Surveillance From 17 February to 12 June 2015, we identified 10,230 suspected cases from the six treatment centers established by MoH. Cases were distributed widely throughout Kampala and neighboring areas (Fig. 1). The epidemic curve of suspected cases suggests that the outbreak started at the beginning of February or perhaps earlier. By the time the outbreak was recognized on 6 February, hundreds of cases had already occurred (Fig. 2). Cases were reported in all five divisions of Kampala: Makindye (32%, 3234), Rubaga (28%, 2828), Kawempe (11%, 1144), Nakawa (6.4%, 656) and Central (4.2%, 428); for 19% (1940) of cases, either no division of residence was identified, or resided outside of Kampala. The attack rate during the outbreak period was highest in Makindye (10/1,000), Rubaga (8.7/1000), and Central (6.5/1000) Divisions. Males had a higher attack rate than females. The attack rate among people in the 15–59 year age group (12/1000) was 6 times higher than among younger (2.0/1000) or older (2.0/1000) persons (Table 1). Case-control study In our hypothesis-generating interviews of patients from the area where the outbreak was first identified, consumption of drinks made with water extracted from unprotected sources and packed in unhygienic conditions was often reported. Of the 33 case-patients we enrolled in the case-control study, 60% were men; the majority of the case-patients (85%) were in the 20–39 year and 9.1% in older age groups. In addition to fever, commonly reported symptoms included abdominal pain (72.72%) and headache (69.69%) (Table 2). We found that 22.58% (7/31) of case patients compared with 2.56% (2/78) of controls usually drank locally packaged water in small plastic bags called “kaveera water†(ORM-H = 8.90; 95% CI = 1.60–49.00); 55% (18/33) of case-patients compared with 19.23% (15/78) of controls drank locally-made passion fruit juice called “butunda†(ORM-H = 4.60; 95% CI: 1.90– 11.00); 31.25% (10/32) of case-patients compared with 16.67% (13/78) of controls usually drank locally-packed pineapple juice called “munanansi†(ORM-H = 2.00; 95% CI = 0.74–5.20); and 15.63% (5/32) of case-patients compared with 8.97% (7/78) of controls usually drank cold millet porridge called “bushera†(ORM-H = 2.80; 95% CI = 0.76–10.00). Workplace as a source of breakfast (ORM-H = 0.25; 95% CI = 0.07–0.93), and workplace as a source of lunch (ORM-H = 0.35; 95% CI = 0.11–1.10) were not significant risk factors for illness. When we compared the proportions of case-patients and controls who drank 0, 1, 2, or 3–4 types of locallymade drinks, we found that case-patients were more likely to drink multiple types of locally-made drinks than controls (Chi-square for linear trend = 14.65, p < 0.001) (Table 3). Laboratory investigation Of 10,230 suspected cases, 3464 (10%) underwent TUBEX® TF testing. Of those, 1038 were positive, representing a positivity rate of 29%. Blood samples from a total of 364 patients (including 20 of 33 case-control study patients) were tested by blood culture and 56 (15%) (including 5 of the 20 case-control study patients tested) yielded S. enterica ser. Typhi. Subsequently, 30 of the 56 S. Typhi isolates from blood cultures were confirmed at the U.S. CDC as S. Typhi. CDC determined the minimum inhibitory concentrations for 17 of these isolates, 5 of which were resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, nalidixic acid, trimethoprim/sulfamethoxazole and had intermediate interpretation to ciprofloxacin. The remaining 12 were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. Environmental investigation One of 3 “kaveera water†samples and the unlabeled bottle of water sold by street vendors contained lactose fermenting bacteria, which are commonly Enterobacteriaceae and associated with fecal contamination. The 2 other “kaveera water†samples contained non-lactose fermenting bacteria, also consistent with fecal contamination, and one isolate was further identified as non-typhoidal Salmonella. The 5 water samples from unprotected springs showed evidence of robust contamination with lactose fermenting bacteria. Lactose non-fermenting colonies from 2 spring water samples were identified as non-typhoidal Salmonella spp. Lactose fermenting bacteria were also detected in 2 of 3 “bushera†samples, 1 of 2 “munanasi†samples, all 3 “munanansi†juice samples, and the “bongo†sample. Additionally, non-typhoidal Salmonella was cultured from 1 “busheraâ€, 1 “munanansiâ€, and 1 passion fruit juice sample. Discussion Our investigation revealed a prolonged and widespread outbreak of typhoid fever that affected thousands of people in all five divisions of Kampala City over several months. Contaminated water from unprotected sources and drinks made with it were the likely vehicles of infection early in the outbreak. Juice and water samples obtained from street vendors and water samples collected from unprotected spring water sources showed evidence of fecal contamination. Although S. Typhi was not recovered from environmental testing, non-Typhi Salmonella were isolated from five street-vended beverage samples from the implicated markets. All 17 isolates of S. Typhi from blood tested at CDC were resistant to nalidixic acid and had intermediate interpretation to ciprofloxacin. It is possible that persons affected by the antibiotic-resistant strains during this outbreak had experienced complications of typhoid fever considering that antibiotic-resistant strains of S. Typhi are associated with more severe form of the illness, complications and death [15]. This outbreak may have started in January 2015 or even earlier; however, it was not recognized until early February because routine clinical and laboratory surveillance systems for typhoid fever were not in place before the investigation. The widespread nature of the outbreak is compatible with a waterborne source. The sudden increase in cases after the start of the investigation was likely due to active community outreach and education about the symptoms of typhoid fever and the availability of prompt, free diagnostic testing and treatment through newly established treatment centers. The gradual decline in cases from mid-March onwards was likely the result of patient treatment and public health interventions including provision of free water chlorination products, sensitization of residents on water treatment, and the establishment of free alternative safe water sources in the most affected communities. Based on the evidence we presented, the Kampala Capital City Authority sealed off all underground water sources and worked with the National Water and Sewerage Corporation to ensure the provision of accessible alternative sources of water to the affected communities. In Uganda, as in many low and middle income countries, definitive diagnostic tests for typhoid fever such as blood culture are usually unavailable, unaffordable, or inconsistently applied [16]. Instead, typhoid fever diagnosis and surveillance often rely on clinical judgment or on the Widal test, which has poor sensitivity and specificity [17]. Moreover, physicians often give presumptive antibiotic and/or antimalarial treatment for febrile illnesses [18, 19] without attempting to determine the etiology. Previous studies have indicated that a significant proportion of febrile illness in Uganda is caused by bacteremia, including invasive non-Typhi salmonellosis and typhoid fever [20]. A more robust approach in these settings could entail periodically identifying persons with febrile illness in the communities and taking blood culture for confirmation [21]. The blood samples could be collected and sent using a specialized transport network to regional laboratory centers around the country where confirmative tests can be performed. This system has been successfully used to improve diagnostic services in early infant HIV/AIDS diagnosis [22]. Sentinel surveillance for febrile illnesses based on blood cultures would accelerate the early identification of outbreaks and implementation of control measures. Waterborne typhoid and paratyphoid fever affect an estimated 27 million people worldwide each year [3]. In developing countries, where safe water and sanitation systems have not been well-established, large-scale typhoid and paratyphoid outbreaks sometimes occur [7, 23–26]. During a previous typhoid outbreak in Kasese and Bundibugyo districts, Uganda, in 2009–2011, which affected 8092 persons, the vehicle of transmission was also found to be unclean water [7]. The current outbreak was likely caused by consuming contaminated water from unprotected ground water sources. Kampala city has more than 200 unprotected ground water sources, most of which serve as unprotected sources of water for economically disadvantaged people in the city such as those in our investigation [27]. Unsafe disposal of excreta and solid waste are significant factors that contribute to contamination of ground water in Kampala [28]. This outbreak investigation highlights the importance of ensuring access to affordable, safe, treated drinking water and improved sanitation and waste management systems for resource-constrained urban populations. Risk factors for typhoid transmission were not assessed later during this outbreak, when foodborne transmission might have become more common. Recurrent contamination of unprotected water sources with S. Typhi likely continued to sustain the outbreak propagation over the course of several months. According to the Uganda Demographic Health Survey 2011 [29], almost 30% of people living in urban areas and more than 60% of those living in rural areas do not treat their water before drinking it. Barriers to safer drinking water include the cost associated with establishing a piped treated water system or purchasing water treatment products for household use and the false perception that naturally occurring water sources could be safe [30]. In the aftermath of outbreaks like this one, public health authorities face 3 possible options: The first option is to do nothing but respond to outbreaks as they occur. Governments in resource-constrained settings often choose this option, leaving the population vulnerable to outbreaks of waterborne diseases including cholera, hepatitis A and E, cryptosporidiosis, shigellosis, and many others in addition to typhoid and paratyphoid fever. The second option is mass vaccination against typhoid fever. A cost-effectiveness evaluation of a hypothetical typhoid vaccination campaign was carried out after the multi-year outbreak of typhoid fever in Kasese District, Uganda, and it was estimated to be highly costeffective [31]. However, vaccination against the many different pathogens that cause waterborne diseases is not possible because vaccines are not yet available for many of them (e.g. cryptosporidiosis, shigellosis, paratyphoid fever, etc.). In addition, typhoid fever vaccines have been shown to have varied levels of effectiveness (from 50 to 95%) and to last for varied lengths of time (from 3 to 10 years) [15]. The third and final option is to improve the water and sanitation systems. Improvement of sanitation, hygiene and clean water supply around the world could avert ≥90% of diarrheal disease episodes annually [32]. In North America and Europe, typhoid fever caused largescale outbreaks from the late 19th through the early 20th century [33, 34]. After improvement of municipal water and sanitation systems in the early 20th century, waterborne outbreaks of communicable diseases including typhoid fever drastically decreased [35–37]. Improved sanitation measures such as having a basic pit latrine or a toilet connected to a septic tank curtail the direct contact between human waste and water or the environment. Yet in 2012, only 33% of the urban population in Uganda had access to adequate sanitation, an increase of only 1% since 1990, and 2% still practiced open defecation [38]. Although improving water and sanitation systems requires a substantial investment by the government, ultimately it is highly cost-effective in the reduction of many waterborne diseases [39]. Strengths and limitations A major limitation of our investigation was that, due to inadequate laboratory capacity to confirm a large number of cases early in the outbreak, and the need to rapidly identify the mode of transmission to inform effective interventions, we included non-laboratory confirmed cases in our case-control study. While a clinical case definition for typhoid fever cases can lead to misclassification, and is not recommended during nonoutbreak situations, during an outbreak such a case definition will often perform well, as measured by good positive and negative predictive values [40]. Also, the information on usual sources of water and food was based on self-reports, which could represent a source ofinformation bias. Another limitation is that data on mortality or on complications such as intestinal perforations were not collected. With over 10,000 cases it is likely that there were intestinal perforations and deaths but no surveillance for those outcomes was done. A study is currently being conducted to assess these severe impacts of this outbreak. In addition, only a few water and juice samples were tested, which could explain why S. Typhi was not isolated during the environmental investigation. Also, water and juice samples were tested using the reagents and procedures that were available in the laboratory for testing clinical specimens for Salmonella, and not more conventional methods for evaluating the potential presence of fecal contamination in these types of samples. Conclusion In conclusion, this investigation revealed a large outbreak of typhoid fever that affected thousands of people in Kampala, Uganda, which appeared to have been caused by consuming contaminated water and local drinks made from it. To prevent future waterborne outbreaks, we recommended that the Kampala Capital City Authority, the MoH, the National Water and Sewerage Corporation, and partners invest in improving access to potable water, and safe sanitation and hygiene facilities [41].

How long was the event?