Epidemiologic investigation
In this outbreak, study population consisted of suspected cases, probable cases and confirmed cases with the following definitions. Suspected cases were defined as company staff with an onset of vomiting or diarrhea (≥once/day) between September 14 and September 25, 2009. Probable cases were defined as those with an onset of diarrhea (≥three times/day) or vomiting (≥twice/day). Confirmed cases were those probable cases that were tested positive for norovirus by reverse transcription-polymerase chain reaction (RT-PCR). In this investigation, we defined diarrhea as having more than one loose or liquid stools per day, or as having more stools than normal for that person. A retrospective cohort study was conducted and possible risk factors were drinking water or food from the cafeteria in this factory. Information on drinking-water preference and frequency, eating in the factory cafeteria or not, hand-washing habits, eating habits, gender, age, workplace, history of contact with a person with diarrhea and/or vomiting were collected through a questionnaire. Drinking-water preference contained the following two categories: drinking water from directly drinkable-water dispensers (DDWDs) or commercial bottled water. This study was approved by the Ethics Committee of Shenzhen Bao’an Center for Disease Control and Prevention (2009010).
Laboratory tests
Samples from 29 water specimens were collected, including 13 from the water tanks on the top of five buildings, nine from drinking-water machines, five from bottled water, and two from the underground reservoir. All samples were transported to Bao’an District Center for Disease Control and Prevention. All water samples were tested according to the Standards for Drinking Water Quality (GB5749-2006), including the detection of total coliforms, thermotolerant coliforms, Escherichia coli, Salmonella, Shigella, Campylobacter, and Yersinia enterocolitica[11]. In addition, we also tested for norovirus and rotavirus.
As we suspected noroviruses were the responsible agent, we began to collect the specimens of patients. A total of 12 stool and 9 rectal swab samples were collected to test for norovirus. These samples were also transported into Bao’an District Center for Disease Control and Prevention, and detected for norvirus and rotavirus.
RNA extraction and amplification
Virus concentrations of water samples were based on positively-charged filters from 1-l samples, as described previously [12]. Approximately 50–80-μg stool samples were weighed, diluted 1:10 in nuclease-free H2O, and then vortexed for 30 s. Samples were clarified by centrifugation at 6,800 × g for 10 min at room temperature. Viral RNA was extracted from 140-μl processed samples using a QIAamp Viral RNA kit (Qiagen, Victoria, Australia), according to the manufacturer’s instructions.
Real-time fluorescence RT-PCR
Real-time fluorescence PCR for rotavirus and norovirus was performed using an ABI 7500 real-time PCR system with a commercial kit according to the manufacturer’s instructions (catalog numbers SA-6261 for rotavirus and SA-6251 for norovirus, Beijing Suoao Biotechnology Company Limited, Beijing, China).
Environmental investigation and exclusion of pollution sources
The factory comprised an area of 350,000 m2, consisting of three buildings (A1, A2, A3) used as workshops, three buildings (A21, A22, A23) used as dormitories for workers, one building (A24) as a canteen, one building (A15) as a kitchen, and one building as a repair room. About 13,000 workers ate breakfast, more than 6,000 ate lunch, and over 11,000 ate dinner at the company cafeteria every day. Water was supplied from the municipal water supply system and stored in an underground reservoir with a capacity of 8,000 tons. Water from the underground reservoir was pumped into water tanks on top of eight buildings (A11, A1, A12, A3, A21, A22, A23, and A15), and then supplied to DDWDs provided at multiple sites in these eight buildings. Each floor of the dormitory buildings had two DDWDs.
The environment around the underground reservoir is shown in Figure 1. Three possible contamination sources are indicated by red circles. The sewer pipe was close to the municipal water supply pipe. To exclude contamination of the municipal supply water system from the sewer pipe, we collected water specimens from the junction between the municipal water supply system and the underground reservoir to test for bacteria and viruses. Secondly, to exclude contamination from the septic tank near building A23, we looked for seepage tracks from the side of the septic tank on the wall of the underground reservoir. Finally, we checked the integrity of the reservoir lid in order to determine whether contaminants had entered the underground reservoir through this lid.
Statistical analysis
The distribution of major symptom in workers was summarized by frequency and percent. A retrospective cohort study was conducted to investigate possible risk factors for acute gastroenteritis among a workshop of employees in the company with outbreak of acute gastroenteritis. Chi-square test were performed using OpenEpi software version 2.3.1 online (http://www.openepi.com/OE2.3/Menu/OpenEpiMenu.htm). Relative risks (RR) and 95% confidence intervals (CI) were calculated.