A Point Source Gastroenteritis Outbreak in a High School Putatively Due to Clostridium perfringens : Timely Investigation Is Everything

Abstract

On May 28, 2021, several gastroenteritis cases were reported among students and staff of a high school on a Greek island. Lunches were delivered daily at the school by a catering company. We conducted a retrospective cohort study. Cases were defined as students or school staff presenting with diarrhea and abdominal pain between May 27 and 29, 2021. We used a self-administered questionnaire to collect the data. In the univariate analysis, we calculated food-specific attack rates (ARs), relative risks (RRs), and confidence intervals (95% CIs). Statistically significant variables (p < 0.05) were included in the multivariable model. Stool samples were collected from students on the same day of notification and were tested for bacteria, viruses, and parasites. The local food safety authority also inspected the school and the catering company premises. The food samples were sent to the reference laboratory for analysis. We received 129 responses from 174 students and staff members (response rate: 74%). Thirty cases were identified (AR, 23%). Of the 11 significant food items in the univariate analysis, the consumption of spaghetti with minced meat on May 27 (odds ratio 53.40, 95% CI: 3.9–736.1, 97% of cases exposed) was independently associated with the onset of gastroenteritis. Clostridium perfringens was detected in two of the three students' stool samples. Six samples from spaghetti with minced meat were positive for C. perfringens and four of those also tested positive for Bacillus cereus. Environmental investigation revealed non-compliances concerning food personnel training and Hazard Analysis and Critical Control Points (HACCP) plan implementation in the food establishment. Timely investigation and samples collection allowed the identification of the probable source of the outbreak. Changes, regarding the reception and distribution of meals in the school, were applied. Recommendations were given to the catering establishment focusing on adequate personnel training and HACCP issues, especially regarding temperature/time controls during all production, handling, and distribution phases.

Introduction

In 2020, at European Union (EU) level, 527 gastroenteritis outbreaks caused by bacterial toxins were reported by 12 Member States (MS).

Clostridium perfringens toxins were reported by 8 MS and by 2 EU non-MS as being the second most commonly cause of foodborne outbreaks (32 outbreaks, 682 cases, 10 hospitalizations, and 2 deaths), following Bacillus cereus (71 outbreaks, 835 cases, 10 hospitalizations, and 1 death) (European Food Safety Authority [EFSA], 2021).

C. perfringens outbreaks are usually associated with the consumption of contaminated and improperly cooked and stored meat or poultry products. These outbreaks usually affect up to hundreds of people in settings such as hospitals, schools, prisons, nursing homes, and institutions or events with catered food (Ameme et al, 2016; CDC, 2022; Domínguez-Berjón et al, 2003; McClane, 2007).

C. perfringens causes a short and mild gastrointestinal illness with symptoms, including diarrhea, nausea, abdominal pain while vomiting, and fever are not frequently reported (Dolan et al, 2016; Heymann, 2008; Kalinowski et al, 2003; Moffatt et al, 2011). Patients rarely seek medical assistance (Ameme et al, 2016; Heymann, 2008). Laboratory confirmation of C. perfringens as the causative agent of a foodborne outbreak is challenging, as it can be randomly found in human and animal stools and is also widespread in the environment (Kalinowski et al, 2003).

On May 28, 2021, the Hellenic National Public Health Organization (NPHO) was informed by the Hellenic Food Authority (EFET)-Regional Directorate of the North Aegean Sea, about a cluster of gastroenteritis cases among students (12–18 years old) and teachers at a high school. The school is located in a small town on a remote north-eastern Aegean Greek island with 86,000 permanent residents and has a limited capacity for specific laboratory testing.

Phone contact with the school principal led to outbreak verification. Based on the initial communications with the principal of the school and the students, apart from school attendance, no other common out-of-school activities had taken place during previous days. Daily hot lunches were delivered at school by a catering company and were consumed inside the classrooms. No children or teachers had severe symptoms to visit the local health care services.

No other outbreaks were reported to be linked to the same catering company. On May 28, 2021, the NPHO started an outbreak investigation. An already established protocol for timely outbreak response in collaboration with local public health authorities and the school's principal was followed. The objectives of this investigation were to identify the size of the outbreak, the etiology and vehicle of transmission to implement appropriate control measures and prevent similar events.

Methods

Epidemiological investigation

Study design, recruitment, and data collection

A retrospective cohort study was performed among all students and staff of the affected high school (126 enrolled students and 48 teachers). A self-administered, semi-structured questionnaire was sent on May 31, 2021, via email, to the school for data collection. Questions focused on demographic characteristics (sex, age, school grade), symptoms' onset, clinical manifestations, hospitalization, and school exposures (consumption of food items and drinks from the school canteen and catered lunch meals from May 24 to May 27, 2021).

Written informed consent was provided by the adults and the students' parents/guardians. All data were anonymized and used only for outbreak investigation.

A case of gastroenteritis was defined as any student or staff member of the high school who developed diarrhea and abdominal pain between May 27 and May 29, 2021. The median time between the symptoms' onset in all identified cases and the consumption of the incriminated meal (incubation period) was estimated in hours. This study was approved by the NPHO Research Ethics Committee. This study was conducted in accordance with the guidelines of the Declaration of Helsinki as revised in 2013.

Data analysis

Food-specific attack rates (ARs) were calculated. A univariate analysis was performed to test for possible associations between food exposures and acute gastroenteritis occurrence by calculating relative risks (RRs) and confidence intervals (95% CIs). Statistically significant associations (p < 0.05) in the univariate analysis were included in the multivariable model. Multivariable analysis was performed using logistic regression with backward elimination. Epi Data 3.5.3 was used for data entry and STATA v16.0 (Stata Corporation, TX) for statistical analysis.

Microbiological investigation of clinical samples

All the cases were recommended to visit the local hospital to submit clinical samples. On the 28th of May, the collected samples were sent to the Central Public Health Laboratory (KEDY) of NPHO to be tested with the method of multiplex PCR (BioFire Film Array Gastrointestinal Panel) for 22 enteropathogens (Enterotoxigenic Escherichia coli [ETEC], Enteropathogenic E. coli [EPEC], Enteroaggregative E. coli [EAEC] Shiga-like toxin–producing E. coli [STEC] with specific identification of E. coli O157, Shigella spp./Enteroinvasive E. coli./[EIEC], Campylobacter spp., Vibrio cholerae, Vibrio spp., Yersinia enterocolitica, Plesiomonas shigelloides, Clostridium difficile [Toxin A/B], Salmonella spp., Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Adenovirus F40/41, Astrovirus, Norovirus GI/GII, Rotavirus A and Sapovirus [genogroups I, II, IV and V]) (Buss et al, 2015).

After this initial testing, and as the clinical picture of the patients was compatible with gastroenteritis due to bacterial toxins, the three stool samples were sent to the Regional Laboratory of Public Health of Thessaly (PEDY), at Larissa, for toxin detection. Unfortunately, at the time only culture and not toxin detection for C. perfringens and B. cereus could be performed.

Environmental investigation

Food sampling and further inspection of the two (2) involved facilities was conducted by EFET-Regional Directorate of the North Aegean Sea. First, on May 28, 2021 food safety officers visited the school to collect samples from implicated meals. The record of the overall management of lunches' distribution, along with any relevant procedure followed by the assigned staff (school teachers) was also conducted. Six (6) whole intact prepacked meals delivered to school on May 27, 2021 (spaghetti with minced meat) were found in the school's refrigerator stored at ∼4°C.

These meals were part of the meal offered the previous day, just before the occurrence of the cases and therefore were all collected for testing. By the time of sampling, all food samples were kept under refrigeration conditions for almost 24 h and sent to PEDY of Thessaly. The food samples were tested for Salmonella spp. (ISO 6579-1:2017) and Listeria monocytogenes (ISO 11290-1:2017) detection. Further, the determination of Total Plate Count (TPC) (ISO 4833-1:2003) and enumeration of the samples' coagulase-positive staphylococci (Staphylococcus aureus and other species) (ISO 6888-01:1999/Amd. 1:2003), E. coli (ISO 16649-2:2001), Total coliforms (ISO 4832:2006), B. cereus (ISO 7932:2004), and sulfite-reducing Clostridium spp. (ISO 15213:2017) was performed.

Due to geographical/location issues, transportation lasted ∼20 h, during which all food samples were kept in a thermobox along with ice packs.

In addition, on May 31, 2021 and June 02, 2021, food safety officers visited the meals' production establishment and conducted inspection. The inspection concerned the premises, the equipment, the standard operational procedures, and Hazard Analysis and Critical Control Points (HACCP) plan along with the operational prerequisite programs. No food sampling was carried out due to the fact that none of the raw materials (i.e., minced meat) used in the implicated meals were found in the establishment.

Results

Epidemiological investigation

The cohort comprised 126 students and 48 teaching staff members. From the cohort population, 129 people completed the questionnaire (response rate: 74%). Of the total number of participants, 98 (76%) were students, 26 (20%) were teachers, whereas 5 (4%) did not complete the respective information. The median age of respondents was 14 years (range: 12–65 years). We identified 30 cases, 26 students (87%) and 4 teachers (13%) who met the case definition (overall AR: 23%).

Sixty percent of the cases were women, and the median age of the cases was 13 years (range: 12–40 years). The AR was high among students in 7th grade (35%), 8th grade (34%), and 11th grade (20%).

The distribution of cases by the date and time of symptoms onset is shown in Figure 1. The outbreak peaked on May 28, 2021, at night (00:00–06:00h). The shape of the epidemic curve suggested a common point source outbreak. Most cases (26, 87%) reported abdominal pain and diarrhea, whereas 19 (66%) reported fatigue. They infrequently reported nausea (8, 27%), myalgia (4, 14%), or vomiting (1, 3%). The median duration of symptoms was 1 day (0.5–3 days). None of the cases was hospitalized. The median estimated incubation time was 14 h (1–21 h). An outlier of 41 h was not included in the estimation of the incubation period.

FIG. 1.

Distribution of cases by date and time of symptom onset, in a high school on a Greek island, May 2021 (n = 21). For nine cases, the exact time of symptom onset was not known and was therefore not included in the epicurve. NPHO, National Public Health Organization.

Table 1 presents the ARs for food items consumed by the students and teachers by three school days during week 21, 2021. Univariate analysis revealed a statistically significant association between disease occurrence and the consumption of spaghetti with minced meat during the lunch served on May 27, 2021, at 13:00 (RR: 50.6, 95% CI: 7.1–359.5).

Table 1.

Results of Univariate Analysis of Food Items Consumed by Students and Teachers in a High School from 24 to 27 May on a Greek North-Eastern Aegean Island, May 2021

Food items	Among exposed			Among unexposed
Food items	Total	Cases	AR (%)	Total	Cases	AR (%)	RR	95% CI
Monday, May 24
Chicken	42	18	42.86	86	11	−12.79	3.35	1.74–6.44
Rice	46	20	43.48	82	9	10.98	3.96	1.97–7.97
Cabbage salad	22	9	40.91	105	20	19.05	2.15	1.13–4.07
Bread	31	12	38.71	97	17	17.53	2.21	1.19–4.10
Apple	34	11	32.35	94	18	19.15	1.69	0.89–3.20
Tuesday, May 25
Burger	56	21	37.50	72	8	11.11	3.38	1.62–7.04
Baked potatoes	56	22	39.29	72	7	9.72	4.04	1.86–8.78
Cabbage salad	18	6	33.33	110	23	20.91	1.59	0.75–3.37
Bread	31	13	41.94	97	16	16.49	2.54	1.38–4.68
Rice with milk	27	8	29.63	101	21	20.79	1.43	0.71–2.85
Wednesday, May 26
Omelette	23	10	43.48	105	19	18.10	2.40	1.29–4.46
Cabbage salad	14	6	42.86	114	23	20.18	2.12	1.05–4.31
Bread	26	12	46.15	102	17	16.67	2.77	1.52–5.05
Rice with milk	21	7	33.33	106	22	20.75	1.61	0.79–3.27
Thursday, May 27
Spaghetti with minced meat	47	29	61.70	82	1	1.22	50.60	7.12–359.54
Feta cheese	17	6	35.29	112	24	21.43	1.65	0.79–3.43
Bread	31	15	48.39	98	15	15.31	3.16	1.75–5.71
Orange	29	9	31.03	100	21	21.00	1.48	0.76–2.87

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

Almost all cases (97%) were exposed to this lunch dish. The sole exposure that was significantly associated with the onset of gastroenteritis in the multivariable analysis was the consumption of spaghetti with minced meat (odds ratio = 53.40, 95% CI: 3.9–736.1) (shown with bold values in Table 2).

Table 2.

Results of Multivariable Analysis of Food Consumed by Students and Teachers in a School Setting, on a Greek North-Eastern Aegean Island, May 2021

Food items	ΟR	Std. Err	z	p > \|z\|	95% CI
Spaghetti with minced meat, May 27, 2021	53.40	71.48	2.97	0.003	3.87–736.13
Rice, May 24, 2021	8.33	10.42	1.70	0.090	0.72–96.56
Baked potatoes, May 25, 2021	0.69	0.77	−0.33	0.738	0.08–6.18
Chicken, May 24, 2021	0.22	0.26	−1.28	0.201	0.02–2.23
Bread, May 27, 2021	3.86	6.13	0.85	0.395	0.17–86.92
Burger, May 25, 2021	1.61	1.59	0.48	0.633	0.23–11.24
Bread, May 26, 2021	3.10	3.49	1.00	0.316	0.34–28.25
Bread, May 25, 2021	0.52	1.08	−0.31	0.753	0.00–29.75
Omelette, May 26, 2021	2.52	2.47	0.95	0.344	0.37–17.21
Bread, May 24, 2021	0.28	0.40	−0.88	0.378	0.02–4.69
Cabbage salad, May 24, 2021	1.22	1.15	0.22	0.828	0.19–7.68

OR, odds ratio; Std. Err. standard error.

Microbiological investigation

Overall, three symptomatic students visited the health care facility to submit stool samples. Of the three stool samples tested using the multiplex PCR, one was positive for EPEC. The other two samples were negative for all enteropathogens tested. From further laboratory investigations of the three stool samples at PEDY of Thessaly, two were positive for C. perfringens detection whereas all were negative for B. cereus detection.

One of the two positive for C. perfringens was the one that had been tested positive for EPEC. From the microbiological analysis of the food samples, the following results were obtained: All six samples displayed TPC between 2.8 × 10⁴ and 9.5 × 10⁸ colony-forming unit (CFU)/g, with suggested HACCP critical limits for ready-to-eat foodstuff <10⁴ CFU/g. All six samples tested displayed concentrations of total coliforms between 10⁴ and 2.5 × 10⁷ CFU/g, with suggested HACCP critical limits <10³ CFU/g.

Two out of six food samples had an E. coli concentration between 2.3 × 10³ and 2.1 × 10⁶ CFU/g, with suggested HACCP critical limits <10 CFU/g. Four out of six samples had B. cereus concentrations between 5.4 × 10⁴ and 4.5 × 10⁶CFU/g and finally all six samples presented concentrations of C. perfringens between 280 and 3 × 10⁶CFU/g, with suggested HACCP critical limits <100 CFU/g.

Environmental investigation

The EFET conducted environmental investigations on two sites. The first one was the school where suspected meals were delivered, whereas the second one was the food establishment where these meals were previously produced.

During the environmental inspection, deficiencies in the reception and distribution of lunch meals, by both the catering company and the receiving committee of the school, were identified. The mass catering establishment did not accurately describe the type of lunch meals in the delivery notes, and the temperature of the meals was not checked on receipt.

Τhe school meal committee could only record the number of meals received. Further, it was not specified whether the time elapsed between the preparation and consumption of the lunch meals at school was <2 h. The absence of written protocols regarding step-by-step quality and safety assurance was the main finding of the inspection of the provision of lunch meals at the school premises. However, there was no evidence suggesting any contribution to the outbreak, since the elapsed time between meals' preparation and consumption is a parameter within the field of responsibilities of the catering provider.

On the other hand, the investigation in the food establishment revealed non-compliances concerning food personnel training and HACCP plan implementation. Food business operator failed to ensure that: (1) personnel was adequately and effectively trained in the application of the HACCP principles according to Regulation (EC) No 852/2004, Annex II, Chapter XII, at all times and (2) preparation and distribution of school meals was incorporated in the scope of existing HACCP plan (article 5 of the above Regulation).

More specifically, what triggered food safety inspectors' interest was mince meat preparation and hot holding chamber's temperature before transportation and distribution. On May 22, 2021, raw mince meat was supplied, marinated, blanched, and frozen in a blast freezer until further cooking took place 5 days later. Then, the spaghetti was cooked and mixed with minced meat, transported, and served at the school.

Nonetheless, no relevant written procedures and records were provided by the catering establishment during investigation. In addition, meals, right after being cooked, were packed in individual portions and transferred into a hot holding chamber. Meals were temporarily stored until all portions were ready to be loaded in the transport vehicles and placed in isothermal boxes.

Although the temperature of the chamber was meant to be 75°C, during the investigation the temperature was ∼55°C. Moreover, part from the same mince meat batch supplied on May 22 was prepared and served on May 25 elsewhere, without any food incidents reported, suggesting that there was a mishandling during the production of the meals or/and their distribution to the school involved.

Communication and measures taken

Recommendations were given to the school's principal, regarding (1) training of the personnel on hygiene and food safety issues, (2) the use of a food temperature control thermometer, (3) the daily storage of food samples under specific conditions, (4) the laboratory control of samples whenever necessary, (5) recording of the procedures, and (6) adherence to specific rules regarding the time and place of food consumption.

The provision of meals to people outside the school should not be allowed (families and students of the co-located high school). Toward that direction, a one-page record form was prepared and given to the school's principal for the school committee's convenience. The above records were monitored by EFET officers until the end of the school period (June 2021).

Regarding the catering establishment, it was recommended: (1) to incorporate the preparation and distribution of school meals in the scope of the existing HACCP plan, and to implement effective record keeping to affirm compliance with the HACCP plan and (2) to ensure that there will be adequate and effective training of personnel in the application of the HACCP principles. The importance of the following actions was emphasized: (1) applying good hygienic practices at all times and especially during freezing and defrosting of mince meat (raw or partially cooked), (2) keeping the temperature of the hot holding chamber high enough to ensure that the temperature in the center of ready to eat meals after cooking will be at least 65°C. Since the end of the investigations, no other food incident occurred involving any of the parties implicated.

Discussion

A foodborne outbreak occurred among high-school students and teachers on a remote Greek island in May 2021.

Based on the results of the conducted retrospective cohort study, consumption of spaghetti with minced meat, offered as school lunch on May 27, was identified as the main risk factor for the occurrence of gastroenteritis symptoms. This finding was strengthened by the results of microbiological analysis of food samples that contained micro-organisms (total coliforms, E. coli, B. cereus, and C. perfringens) in quantities that present an unacceptable risk for human health.

C. perfringens was the most probable etiological agent of this outbreak as: (1) it was isolated in both human and food samples, (2) the documented clinical manifestations were more compatible with C. perfringens infection rather than EPEC or B. cereus infection (McClane, 2007, Heymann, 2008, Hawker et al, 2012;), and (3) meat and meat products have been documented as the vehicle of foodborne outbreaks caused by C. perfringens (EFSA, 2021). However, the etiological agent was not confirmed, as laboratory investigations of C. perfringens toxins in clinical and food samples were not performed due to lack of laboratory capacity.

C. perfringens is a common causative agent of foodborne outbreaks globally, even though these outbreaks are often underreported because of the mild nature and the relatively short duration of symptoms (Hawker et al, 2012). There are a few references of such outbreaks in the literature (Ameme et al, 2016; Domínguez-Berjón et al, 2003; Leung et al, 2016; Rinsky et al, 2015; Simone et al, 2013). In Greece, C. perfringens was identified as the causative pathogen in one out of 403 foodborne outbreaks reported and investigated by the NPHO during 2004–2020 (Mellou et al, 2019).

To improve the detection of outbreaks caused by C. perfringens, emphasis should be placed on surveillance, rapid response, acquisition of detailed history on clinical manifestations, and targeted laboratory analysis. In this outbreak, quick response enabled the isolation of C. perfringens in both food and stool samples.

Although provision of catered meals in schools is not a common practice in Greece, our investigation identified a lack in the implementation of food safety measures in such settings. The importance of proper investigation and immediate implementation of public health measures in case of foodborne outbreaks in school settings, where many people can be affected within a short time, has been documented (Lee and Greig, 2010).

In this outbreak, recommendations mainly referred to the food production establishment (catering) and focused on adequate personnel training and HACCP issues, especially regarding temperature/time controls during all production, handling, and distribution phases. The main limitation of environmental investigation of the catering establishment was that it did not robustly conclude on the exact points in food preparation/storage/transportation that led to the outbreak. Therefore, only general recommendations were provided.

Conclusions

Timely investigation and interdisciplinary collaboration allowed for the rapid identification of the probable vehicle and causative agent of this outbreak. However, laboratory capacity for the detection of bacterial toxins in the country and implementation of food safety measures in school settings should be strengthened. Routine inspections of catering companies for assurance of the implementation of HACCP principles is a prerequisite for the prevention of similar outbreaks in closed settings.

Footnotes

Acknowledgments

The authors acknowledge the work of colleagues from the Central Public Health Laboratory, Athens, the Regional Public Health Laboratory of Thessaly, the director of the Laboratory of the island Hospital, and the Principal and the Vice Principal of the high school.

Authors' Contributions

Writing original draft: S.P.; Investigation: S.P., T.S., A.C., M.A.K., V.A.M., S.S. D.B.; Writing—review and editing: S.P.; K.M., Α.C., T.S., S.S., M.A.K; Supervision: T.S., K.M.

Disclaimer

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the institutions with which the authors are affiliated.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the NPHO, Athens, Greece. This outbreak investigation received no grants from any funding agency in the public, commercial, or not-for-profit sectors.

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