Epidemiology of Campylobacter jejuni Outbreak in a Middle School in Incheon, Korea

Outbreak epidemiology

A retrospective cohort study was conducted among students, teachers, and food handlers who were exposed to canteen food in the affected middle school. Self-administered questionnaires were distributed to these individuals from July 6-8 and information was collected on demographic details, symptoms, time of onset and duration of illness, and individual canteen food items consumed from July 1-3. A case was defined as a school member who consumed canteen food from July 1-3 and who exhibited abdominal pain accompanied by diarrhea and/or tenesmus and/or headache from July 3-6, 2009. The average incubation period and the common point of exposure were estimated using an established incubation period model (7).

As part of the investigation, the catering kitchen was examined including specimens taken from utensils, and interviews were conducted with kitchen staff about food handling practices and illness. Preserved foods, drinking water and kitchen utensil specimens were collected and examined at the public health office. Symptomatic people and food handlers were asked to submit stool samples for standard bacteriological, virological, and protozoan examinations (Table 1). Food and water specific attack rates (ARs), relative risk (RR), 95% confidence interval (95% CI) were calculated, and ARs were compared among groups or grades using chi-squared test. The association between consumption of drinking water or food and the risk of disease was determined using chi-squared or Fisher's exact tests. Upon menu analyses on each day (July 1-3), cases that had occurred before lunch were excluded. Statistical analyses were made using SPSS for Windows (version 10.0; SPSS Institute, Chicago, IL, USA).

Table 1
Examination of environmental specimens and rectal swabs

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Microbial identification and pulsed-field gel electrophoresis (PFGE)

Each sample (stool or rectal swab) was planted to Preston broth (Oxoid, Basingstoke, UK) and incubated at 42℃ for 48 hr under microaerobic conditions (5% O₂, 5% CO₂, 85% N₂). The sample was then streaked onto Campylobacter blood free selective agar (Oxoid). One presumptive Campylobacter isolate from each sample was identified to the species level on the basis of Gram staining, catalase and oxidase production, hippurate hydrolysis and API Campy system (Biomerieux, Marcyl'etoile, France). Further identification was performed with PCR using primer sets specific to hipO gene in C. jejuni. Template DNA for PCR was prepared by boiling method. PCR was performed using a C. jejuni kit (Rapigen, Gunpo, Korea) in accordance with the manufacturer's instruction.

A total of 31 Campylobacter spp. were analysed by PFGE using the restriction enzyme SmaI. The isolates were suspended in 2 mL of cell suspension TE buffer (100 mM Tris, 100 mM EDTA, pH 7.5). A 200 µL aliquot of adjusted cell suspensions and an equal volume (200 µL) of melted 1.2% SeaKem Gold agarose were mixed by pipetting. The agarose cell suspension mixture was dispensed into the wells of plug molds (Bio-Rad Laboratories, Hercules, CA, USA) and solidified at 4℃ for 5 min. The plugs were transferred to 2 mL tubes containing 1.5 mL of ES buffer (0.5 M EDTA, 1% sodium-lauroyl-sarcosine) and 40 µL of proteinase K (20 mg/mL). Lysis was allowed to proceed for 90 min at 55℃ in an orbital shaker water bath. After lysis, the plugs were washed five times with plug wash TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5).

A 2 mm wide slice from each plug was cut with a single-edge razor blade and transferred to a tube containing 100 µL of the restriction enzyme mixture (40 unit SmaI 1 µL, 100 BSA 1 µL, buffer 10 µL, DW 88 µL). The plug slices were incubated at 25℃ for 4 hr. The electrophoresis conditions consisted of an initial switch time of 6.76 sec and a final switch time of 35.38 sec (gradient of 6 V/cm and an included angle of 120). The gels were electrophoresed for 18 hr, depending on the equipment used (CHEF Mapper, Bio-Rad), in 0.5× TBE. After the electrophoresis run was finished, the gels were stained with 500 mL of ethidium bromide solution (50 µg/mL), and the band pattern was observed under UV illumination. Matching and dendrogram UPGMA (unweighted pair group method with averages) analysis of the PFGE patterns was performed using the Dice coefficient with a 1.0% tolerance window.

Epidemiologic findings

Of the 921 school members (854 students, 48 teachers, 11 food handlers, eight other employees) who consumed canteen food, 791 (85.8%) completed the questionnaire. Of these 791 people (760 males, 31 females), 738 (93.2%) were students, 38 (4.8%) were teachers, and 11 (1.3%) were food handlers. Ninety-two school members met the case definition, representing an overall AR of 11.6% (92/791). AR did not differ significantly among students, teachers, and food handlers, but was significantly higher in second-grade students than students in other grades (P≤0.01) (Table 2).

Table 2
Attack rate by group in a middle school in Incheon, Korea, 2009

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Day of illness onset for the 92 cases is shown in Fig. 1. The epidemic curve showed characteristics of a point source outbreak. The canteen food and drinking water were provided during lunch time (12:00-13:00). The first patient developed symptoms beginning at approximately 1:30 a.m. on July 3. After a slight increase beginning on July 3, the number of cases rose sharply to a distinct peak on July 4 and decreased exponentially over the subsequent 2 days. The average incubation period for this outbreak was 2.13 days (95% CI: 0.66-6.81 days). The date of exposure was considered to be July 1. The main symptoms were abdominal pain (100%) and diarrhea (80.4%). Tenesmus (62%) and headache (48.9%) were less frequent (Table 3).

Fig. 1
The epidemic curve by symptom onset date in a middle school.

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Table 3
Prevalence of symptoms in cases^* in a middle school in Incheon, Korea, 2009

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Laboratory findings

A total of 77 stool samples were provided from 66 symptomatic people and 11 food handlers. Of the 66 patients who provided stool samples (Table 4), 31 samples (46.9%) were positive for C. jejuni. All these samples came from students. Among these 66 patients, only 36 completed the questionnaire; of the 36 respondents, 10 were positive for C. jejuni. Five (7.5%) samples were positive for enteropathogenic Escherichia coli (EPEC). EPEC was simultaneously detected in two students who were positive for C. jejuni. The small number of EPEC cases effectively excluded the bacterium as the causative pathogen of the outbreak.

Table 4
Result of microbiological examinations

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Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA isolated from patients revealed three patterns: DBRS16.001 (n=29 strains), DBRS16.002 (n=1), and DBRS16.003 (n=1). The DBRS16.001 pattern shared 92.3% and 85.5% similarity with the DBRS16.002 pattern and DBRS16.003 pattern, respectively (Fig. 2), consistent with a single source of infection in the outbreak.

Fig. 2
Results of PFGE analyses.

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The culture of drinking water and the other environmental specimens showed negative results. Only seven preserved foods were obtained (Korean cabbage kimchi, boiled barley rice, potato and hot pepper paste soup, grilled scabbard fish with curry, steamed sesame leaf, diced radish kimchi, yogurt). The remainder had already been discarded by school officials prior to the investigation. Excluding Korean cabbage kimchi, the foods on July 2 were not kept despite the legal preservation period.

Statistical analyses of food and water

Upon menu analysis, consumption of grilled scabbard fish with curry on July 3 was significantly associated with illness (RR=2.39). The other food items were not significantly associated with illness (Table 5). Based on cross-classification analysis of chicken soup with ginseng and grilled scabbard fish with curry, consumption of both food items was associated with a higher AR than consumption of either food item alone (Table 6).

Table 5
Relative risk of suspected foods

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Table 6
Attack rate by chicken soup with ginseng and grilled scabbard fish with curry

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Environmental investigations

The school food service facility was located between the main building and an annex building. The facility housed its own water supply and was equipped with five water purifiers. The main building had a water purifier on the first and third floor, and one in the teachers' office. The annex building had a water purifier on the third floor. The annex building also housed second-grade classrooms, with the first- and third-grade classrooms located in the main building. The water supplies were used in the food processing but purified water was not used. The original water for the water purifiers was supplied from water supplies. Prior to the July 6 illness outbreak, the purifier filters had all been replaced on May 29 with newly-purchased filters (as verified by the purchase receipts). Water samples had been collected from the water purifiers and tested on June 12 by a contracted inspection business; total coliform counts for all samples were zero.

The residual chlorine concentration rates of the water pipes in the bathrooms in the kitchen, main building, annex building and outside the building were measured; the concentration exceeded 0.4 ppm. There was no evidence of underground water supplies in the water supplies and drainage tax receipts during the last three months. Also, a 30 ton sewage septic tank was located outside the kitchen at distance of about 5 meters. It had been sterilized on June 19 by a service company. The decontamination cleaning certificate and photographs of the cleaning operation were examined.

Additional investigations

Considering laboratory results, chicken soup with ginseng prepared and consumed on July 1 was considered to be the infection source because handling and eating undercooked poultry have consistently been shown to be important risk factors in foodborne illness. The investigation team members revisited the food service facility on July 23, 2009 and performed a thorough investigation of the cooking process and the food ingredients of chicken soup with ginseng. The raw chickens used in the chicken soup with ginseng were supplied frozen (-6℃) in the morning of July 1. The chickens had been slaughtered and processed on June 29. They were deemed acceptable for use in food until July 9 if kept in refrigerated.

The preparation pathway for the chickens used on July 1 was as follows. The raw chickens were stored in a meat refrigerator immediately after being inspected. About 1 hr later, they were cleaned at the meat sterilization sink and transported on meat carts to a couple of large iron pots. All the chickens were boiled in the pots along with jujubes, garlic, and ginseng. An estimated 460 carcasses were simultaneously boiled in each pot. While being boiled, each chicken carcass was removed from the pot and glutinous rice was added into the pot. The central temperature of the chicken or the soup was not recorded. All the prepared chicken carcasses were put into eight food distribution containers and stored in a heating cabinet. Only one food handler was involved in the process of cooking and distributing chicken soup with ginseng. The food handler was not cooking other menus. Other vegetables were cleaned at a vegetable sink and cuttlefishes were cleaned at a fish sink. All the raw chickens had been supplied by company A. Among other schools supplied by the company A, there were no cases of food poisoning. There were no recorded food hygiene regulation violations by company A.

Cuttlefish salads were provided together with the chicken soup with ginseng. The cuttlefishes used in the cuttlefish salads were supplied in a refrigerated state after being handled by company B. Their acceptable period of use was until December 30, 2009. Company B also supplied eight other schools with the same products and no food poisoning occurred in these schools. The ingredients of the grilled scabbard fish with curry prepared on July 3 were curry, soybean oil, fry powder, and frozen scabbard fish. The frozen scabbard fishes were imported from China. Their acceptable period of use was until January 2, 2010. Their core temperature was recorded as 92-98℃. No cases of food poisoning were recorded at the other schools supplied with frozen scabbard fish by the same company. The company was free of food hygiene violations.