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

doi:10.21203/rs.2.10129/v1

Download PDF

Research article

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

https://doi.org/10.21203/rs.2.10129/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 29 Oct, 2019

Read the published version in BMC Infectious Diseases →

You are reading this older preprint version

Read the latest preprint version →

Background

On 4 September 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. At least 209 affected students developed the symptoms of diarrhea and vomiting. Investigations were conducted to identify the agent and source of this outbreak, and to implement controls to prevent further transmission.

Methods

A retrospective cohort study was conducted among the school students and staff in order to test hypothesis that food served at the school canteen. We collected information on demographics, refectory records at the canteen and contact to ill persons or vomit, using a uniform epidemiological questionnaire. Risk ratios (RR) and 95% conﬁdence intervals (CI) were calculated. Stool specimens of cases and canteen employees, food served, water, and environmental swabs were investigated by laboratory analysis.

Results

We identiﬁed 209 cases(including 28 laboratory-confirmed cases) that occurred from August 29 to September 10. All cases were students, and the median age was 20 years, 52% were male. The outbreak lasted for 13 days, and peaked on September 5. Consumption of Drinks window and Rice flour window on 1 September(RR: 3.4, 95% CI: 1.5-7.8, and RR: 7.6, 95% CI: 2.8-20.2), Rice flour window and Fish meal window on 2 September(RR: 4.0, 95% CI: 1.2-13.6, and RR: 4.6, 95% CI: 1.7-12.5), Moslem meal window on 4 September(RR: 2.7, 95%CI: 1.3-5.4), Barbeque window on 5 September(RR: 3.0, 95%CI: 1.2-7.0) were independently associated with increased risk of disease within the following 2 days. Rectal swabs or stool samples from twenty-five cases and ten canteen employees were positive for norovirus GI.6. Five cases were positive for Bacillus cereus, among them, two stool samples were detected by a mixed infection of Norovirus and 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 consumption of high-risk windows was the source of this outbreak. The high rate of infection of both pathogens was exacerbated by unhygienic manipulation and Inappropriate hygiene practices in canteen employees.

Infectious Diseases

Foodborne

Gastroenteritis

norovirus

Bacillus cereus

Outbreak

Cohort study

Norovirus is considered the major cause of acute gastroenteritis among worldwide[1]. Norovirus is highly infectious pathogens that can cause relatively severe disease including vomiting and diarrhea with acute onset. Symptoms usually last for 1-3 days but can persist longer in young, old, and immunocompromised patients. The average incubation period ranges from 12-48 hours[2]. Infections and outbreaks are usually more common in cooler or winter months. Norovirus is transmitted via the faecal-oral route primarily through close contact with an infected person, contact with consumption of contaminated food, water, or environmental surfaces. From 1996 to 2000, 348 norovirus outbreaks were reported in the United States, foodborne accounted for 47%[3]. While 163 norovirus outbreaks were reported in Japan, foodborne accounted for 58.13% from 2001 to 2005[4]. In foodborne norovirus outbreaks for which reported the source of contamination, 70 % were caused by infected food handlers[5,6].

On the other hand, Bacillus cereus is ubiquitous in nature, such as on plants, on 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[7]. 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[8,9], produced during vegetative growth of Bacillus cereus in the small intestine [10], and incubation period ranges from 2-36 hours[2]. While the emetic toxin is produced by growing cells in the food [7]，and incubation period ranges from 8-16 hours[2]. 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 4 September 2018, a boarding school in the Shunyi District of Beijing, China reported an outbreak of acute gastroenteritis. The boarding school had 5043 students. A total of 20 persons had fallen ill on 4 and 5 September with vomiting and diarrhoea. Food from the school canteen served was considered the source of this outbreak. Retained food specimens taken from the school canteen tested positive for Bacillus cereus on 6 September. Meanwhile, Stool samples obtained from 7 students tested positive for norovirus by real-time RT-PCR. To provide effective control measures, we surveyed the outbreak to verification of additional cases, source of infection, vehicle for infection, and mode of transmission.

Study design

When the outbreak was reported, we launched an investigation immediately. The investigation was in response to a public health emergency. Chinese food safety law requires the investigation department to have the right to find out from relevant units and individuals about the situation related to the accident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus it 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 and obtained a part of signed questionnaires. Part of subjects was obtained 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.

Case definition

The investigated subjects included any person 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-to-face questionnaire or by telephone.

Cohort study

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 1 September to 5 September, exposure to high risk foods or patients was most likely to explain the symptoms that occured from 1 to 7 September (assuming an incubation period of 2-48 hours). Exposures prior to 31 August 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 two 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 and analyzed by SPSS 12.0. p-Value was two-sided and p<0.05 was considered statistically signiﬁcant.

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 terminal water and source water. For food and biological samples, we used reverse transcription olymerase chain reaction (RT-PCR) to detect norovirus, and intestinal bacteria including Bacillus cereus, Salmonella, Staphylococcus aureus, Proteus spp., Vibrio parahaemolyticus and pathogenic Escherichia coli by culture. Total bacterial count, total coliforms and thermotolerant coliforms were detected in water samples.

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 hours 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 two 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 two days. We didn't find any high-risk stalls on September 3 (Table 1).

Laboratory inspection

Among 35 samples of rectal swabs or feces from students, 25 samples were positive for norovirus GI.6 (positive rate 71.4%). Bacillus cereus was detected in 5 fecal specimens, and norovirus was also detected in 2 fecal specimens at the same time. 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. 7 environmental samples and 6 drinking water samples 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 meters 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).

The outbreak of acute gastroenteritis, which affected 4.1% of the population of a boarding school, was likely caused by norovirus and Bacillus cereus. 93.8% of the cases occurred in 7 days from September 1 to 7, and the outbreak lasted almost two weeks. The epidemic curve showed that the onset time was concentrated, which strongly indicated that the outbreak was a continuous exposure pattern rather than a human-to-human contact 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, we also established daily cohort and calculated personnel exposure in each stall every day. Finally, we got multiple high-risk stalls, 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[11], 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. 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 [12-15]. Since 1999, the most prevalent genotype in mainland China has been GII.4, accounted for 64% of all detected genotypes[16]. 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 rare[17]. The outbreak was caused by norovirus GI.6. 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. Before the outbreak, in addition to food-borne infections such as eating high-risk foods (such as rice flour and fish meal), canteen employees might also be infected through fecal-oral route, spilled vomit, or physical contact with contaminated environment or aerosols. 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 epidemic situation, 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. The obvious difference in clinical manifestations between Bacillus cereus (diarrheal-type) and noruvirus indicated that Bacillus cereus infection was likely to be a part of the cause of the outbreak.

Based on the experience of the epidemic situation, 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 epidemic. 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. Moreover, supervision department of food safety had more concerns about impact of the epidemic situation and worried about being held accountable, so they had poor collaboration with the Disease control department involved in the investigation. These causes 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. Because the food handlers of the high-risk stalls were isolated or dismissed, the information on food manufacture and sale could not be obtained. This mad 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 the two pathogens infect canteen employees and spread due to their unhygienic operation. Student consumption in these high-risk stalls was the root cause of the outbreak. To determine the possible source of infection and implement targeted intervention measures, It was of great value to start epidemiological investigation in time.

Abbreviations

Not applicable.

Acknowledgements

We would like to thank the staff of the school involved in this outbreak for their help with collection of data for this study. Genuine thanks to the staff of the center for research and superior departments for guide.

Funding

Not applicable.

Availability of data and materials

The data that supports the conclusion of this article is available in the Figshare repository https://figshare.com/articles/dataset_xlsx/7945094.

Authors’ contributions

DWC analyzed the data and drafted the manuscript for the study. DW contributed to the design of the questionnaire. YJL, JCL, XFL, PG, GXZ and WZZ were involved in data collection. HBJ and YL tested the specimens. The manuscript was guided and reviewed by YZ, XCM, HLM and LJZ. All the authors read and approved the final manuscript.

Ethics approval and consent to participate

The investigation of the epidemic was carried out by public health agencies as the legally authorized mandate. Chinese food safety law requires the investigation department to have the right to find out from relevant units and individuals about the situation related to the accident, and collect relevant information and samples. The relevant units and individuals shall cooperate with them and shall not refuse. Thus, it was a minimum risk study that was not subject to the ethical approval of the institutional review committee.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Ahmed SM, Hall AJ, Robinson AE, Verhoef L, Premkumar P, Parashar UD, Koopmans M, Lopman BA. Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis. Lancet Infect Dis. 2014 Aug;14(8):725-730. doi: 10.1016/S1473-3099(14)70767-4.

World Health Organization. Foodborne disease outbreaks: guidelines for investigation and control. Geneva: WHO,2008:45-46.

Thornton AC, Jennings C, Karen S. Norov iruses: agentsin out-breaks of acute gastroenteritis[J]. DMR;2004;2(1):4-9.

Noda M, Fukuda S, Nishio O. Statistical analysis of attack rate in norovirus foodborne outbreaks. Int J Food Microbiol. 2008 Feb 29;122(1-2):216-220. doi:0.1016/j.

Hall AJ, Wikswo ME, Pringle K, Gould LH, Parashar UD. Vital signs: foodborne norovirus outbreaks - United States, 2009-2012. MMWR Morb Mortal Wkly Rep. 2014 Jun 6;63(22):491-5.

Moe CL. Preventing norovirus transmission: how should we handle food handlers? Clin Infect Dis. 2009 Jan 1;48(1):38-40. doi: 10.1086/594119.

Kramer, J.M. and Gilbert, R.J. Bacillus cereus and other Bacillus species. In: Foodborne Bacterial Pathogens (Doyle, M.P., Ed.), pp. 21-70. Marcel Dekker, New York.

Beecher DJ, Wong AC. Tripartite hemolysin BL from Bacillus cereus. Hemolytic analysis of component interactions and a model for its characteristic paradoxical zone phenomenon. J Biol Chem. 1997 Jan 3;272(1):233-9.

Lund T, Granum PE. Comparison of biological effect of the two different enterotoxin complexes isolated from three different strains of Bacillus cereus. Microbiology. 1997 Oct;143 ( Pt 10):3329-3339.

Granum, P.E. Bacillus cereus and its toxins. J. Appl. Bacteriol. Symp. Suppl. 76, 61S-66S.

Nguyen GT, Phan K, Teng I, Pu J, Watanabe T. A systematic review and meta-analysis of the prevalence of norovirus in cases of gastroenteritis in developing countries. Medicine (Baltimore). 2017 Oct;96(40):e8139. doi: 10.1097/MD.

Zhang S, Chen TH, Wang J, Dong C, Pan J, Moe C, Chen W, Yang L, Wang X, Tang H, Li X, Liu P. Symptomatic and asymptomatic infections of rotavirus, norovirus, and adenovirus among hospitalized children in Xi'an, China. J Med Virol. 2011 Aug;83(8):1476-84. doi: 10.1002/jmv.

Gallimore CI, Cubitt D, du Plessis N, Gray JJ. Asymptomatic and symptomatic excretion of noroviruses during a hospital outbreak of gastroenteritis. J Clin Microbiol.2004 May;42(5):2271-4.

Vinjé J. Advances in laboratory methods for detection and typing of norovirus. J Clin Microbiol. 2015 Feb;53(2):373-81. doi: 10.1128/JCM.

Vega E, Barclay L, Gregoricus N, Shirley SH, Lee D, Vinjé J. Genotypic and epidemiologic trends of norovirus outbreaks in the United States, 2009 to 2013. J Clin Microbiol. 2014 Jan;52(1):147-55. doi: 10.1128/JCM.

Yu Y, Yan S, Li B, Pan Y, Wang Y. Genetic diversity and distribution of human norovirus in China (1999–2011). Biomed Res Int. 2014;2014:196169. doi: 10.1155/2014/196169.

Bruggink LD, Oluwatoyin O, Sameer R, Witlox KJ, Marshall JA. Molecular and epidemiological features of gastroenteritis outbreaks involving genogroup I norovirus in Victoria, Austra-lia, 2002–2010. J Med Virol. 2012 Sep;84(9):1437-48. doi: 10.1002/jmv.

Leshem E, Barclay L, Wikswo M, Vega E, Gregoricus N, Parashar UD, Vinjé J, Hall AJ. Genotype GI.6 Norovirus, United States, 2010–2012. Emerg Infect Dis. 2013 Aug;19(8):1317-20. doi: 10.3201/eid1908.

Granum PE, Lund T. Bacillus cereus and its food poisoning toxins. FEMS Microbiol Lett. 1997;157(2):223-228. doi: 10.1111/j.

Additional file 1: The self-compiled case questionnaire used in this survey. The main purpose of this questionnaire is to obtain the basic situation of case and the related risk factors of the outbreak, such as meal situation, contact with the case, etc. The contents of the questionnaire can be obtained in the Figshare repository https://figshare.com/articles/Additional_file_1/8015762.

Table 1 Daily attack rates and risk ratios of gastroenteritis among students having meals on high-risk stalls of the school canteen, September 1–5.

Date	High-risk stalls	Exposed			Non-exposed
Date	High-risk stalls	Total	Cases	AR%	Total	Cases	AR%	RR	95% CI	P
September 1	Drinks	299	13	4.3	777	10	1.3	3.4	1.5-7.8	0.002
	Rice flour	48	6	12.5	1028	17	1.7	7.6	2.8-20.2	0.000
September 2	Rice flour	53	3	5.7	1826	26	1.4	4.0	1.2-13.6	0.046
	Fish meal	81	5	6.2	1798	24	1.3	4.6	1.7-12.5	0.003
September 4	Moslem meal	152	9	5.9	3468	77	2.2	2.7	1.3-5.4	0.008
September 5	Barbeque	152	6	3.9	3059	41	1.3	3.0	1.2-7.0	0.023

AR, attack rate; RR, risk ratio; CI, conﬁdence interval.

Download PDF

Journal Publication

published 29 Oct, 2019

Read the published version in BMC Infectious Diseases →

Editorial decision: Major revision
02 Sep, 2019
Review #2 received at journal
20 Aug, 2019
Reviewer #2 agreed at journal
09 Aug, 2019
Reviewer #3 agreed at journal
09 Aug, 2019
Review #1 received at journal
02 Aug, 2019
Reviewer #1 agreed at journal
16 Jul, 2019
Reviewers invited by journal
15 Jul, 2019
Editor assigned by journal
19 Jun, 2019
Submission checks completed at journal
04 Jun, 2019
Editor invited by journal
04 Jun, 2019
First submitted to journal
24 May, 2019

You are reading this older preprint version

Read the latest preprint version →

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

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Methods

Study design

Case definition

Case ﬁnding

Cohort study

Laboratory and environmental investigations

Results

Descriptive epidemiology

Analytical epidemiology

Laboratory inspection

Environmental hygiene investigation

Discussion

Conclusion

Declarations

Abbreviations

Acknowledgements

Funding

Availability of data and materials

Authors’ contributions

Ethics approval and consent to participate

Consent for publication

Competing interests

References

Additional file

Tables

Status:

Journal Publication

Version 1