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 hours). 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 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 v2010 (Microsoft) and analyzed by SPSS v25.0 software (SPSS Inc., Chicago, IL, USA). p-value was two-sided and p<0.05 was considered statistically signiﬁcant.
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.
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.
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 579bp and 570bp 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).