An outbreak of norovirus infection linked to oyster consumption at a UK restaurant, February 2010

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1 Journal of Public Health Advance Access published October 27, 2010 Journal of Public Health pp. 1 7 doi: /pubmed/fdq089 An outbreak of norovirus infection linked to oyster consumption at a UK restaurant, February 2010 Kenneth Baker 1, Jill Morris 1, Noel McCarthy 1, Luisa Saldana 1, James Lowther 2, Andrew Collinson 3, Michael Young 3 1 Thames Valley Health Protection Unit, Centre for Radiation, Chemical and Environmental Hazards, Health Protection Agency, Chilton, Didcot, Oxon OX11 0RQ, UK 2 European Community Reference Laboratory, Centre for Environment, Fisheries and Aquaculture Science, The Nothe, Barrack Road, Weymouth, Dorset DT4 8UB, UK 3 Wycombe District Council Environmental Health, Queen Victoria Road, High Wycombe, Buckinghamshire HP11 1BB, UK Address correspondence to Jill Morris, jill.morris@hpa.org.uk ABSTRACT Background We present the investigation of an outbreak of gastroenteritis at a UK restaurant incorporating both epidemiological and microbiological analysis. Methods Structured postal questionnaires were sent to 30 diners who ate at the restaurant during the outbreak period (5 7 February 2010). Stool specimens collected from staff and diners were submitted for bacterial culture and norovirus testing, and 15 Pacific oysters (Crassostrea gigas) from the batch served during the outbreak period were tested for norovirus. Results A strong association was observed between illness and oyster consumption (odds ratio undefined, confidence interval: 11.7 to infinity, P ¼ ). Multiple different sequences of norovirus RNA were present in both stool and oyster specimens, typical of a shellfish origin. Several contemporaneous norovirus outbreaks throughout the UK were linked to oysters, particularly, though not exclusively, those sourced from Carlingford Lough in Ireland (as in this study), which were subsequently withdrawn from distribution. Conclusion Despite the risk to human health, there is significant uncertainty surrounding the quantitative correlation between oyster norovirus levels and consumer illness. Continued research should help further our understanding of this crucial correlation and identify ways in which viral depuration of oysters can be enhanced. Keywords norovirus, oyster, outbreak, case control study Introduction Norovirus, a member of the Caliciviridae family of singlestranded RNA viruses, is the most common cause of infective gastroenteritis outbreaks worldwide. 1 In 2009 there were approximately 7000 laboratory-confirmed norovirus infections reported to the Health Protection Agency (HPA), 2 though under-reporting of infection suggests that the true incidence is likely to be at least 1% of the population per year. 3 The incidence of norovirus infection is higher during winter, an observation that has led to the colloquialism winter vomiting disease. 4 Norovirus causes a self-limiting infection, with symptoms of vomiting, diarrhoea, abdominal cramps and myalgia of typically 2 3 days duration. 1 Norovirus commonly causes outbreaks of gastroenteritis in closed settings such as hospitals, residential homes and schools where close contact results in enhanced transmission. 1 Outbreaks can also be food borne, usually as the result of infection (including asymptomatic carriage) in a food handler. 5 However, certain foods can themselves be the source of norovirus, especially frozen raspberries 6 and shellfish. 7 Oysters in particular have the propensity to Kenneth Baker, Public Health F2 Doctor Jill Morris, Consultant in Communicable Disease Control Noel McCarthy, Consultant in Communicable Disease Control Luisa Saldana, Clinical Surveillance Officer James Lowther, Research Scientist Andrew Collinson, Divisional Environmental Health Officer Michael Young, Environmental Health Officer # The Author 2010, Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. 1

2 2 JOURNAL OF PUBLIC HEALTH concentrate norovirus infection when harvested from sewage-contaminated waters, and norovirus outbreaks associated with oyster consumption have been well documented worldwide On the 10 February 2010, the HPA was notified of illness in diners who had suffered from diarrhoea and vomiting after eating at a restaurant on 5 February. This restaurant has approximately 100 covers in two sittings per day, with diners able to select dishes from a la carte or bar menus. The Health Protection Unit convened a formal outbreak control meeting on 11 February 2010, with representatives from the local authority, primary care trust and hospital microbiology teams invited to attend. The aims of the outbreak investigation were to ascertain the extent of the outbreak, understand the source and causative agent of the infection and take necessary action where indicated to halt the spread of infection and help prevent further outbreaks occurring at the premises in the future. Methods Epidemiological investigation The restaurant management was initially contacted by a party of six diners who ate at the restaurant on 5 February Five of these diners became unwell with diarrhoea and vomiting; all except the asymptomatic individual ate oysters. The restaurant then directly contacted six further diners (three couples) who ate on the same day, of whom five consumed oysters and subsequently became unwell. At this point the management alerted the local authority environmental health department who worked with the HPA to investigate the outbreak. Restaurant records identified approximately 300 diners who ate at the restaurant over the weekend period from 5 to 7 February The restaurant management agreed to provide contact details of diners in parties which included at least one person who consumed oysters, however declined to provide further information on other parties. Structured postal questionnaires were sent to all 30 diners for whom contact information was provided. This questionnaire asked individuals to identify which foods from the restaurant were consumed as well as information on the nature of their illness, if any. Initial descriptive analysis was undertaken to describe the population of those affected and pattern of illness. Questionnaire data were used as the basis of a case control study, with case and control definitions as described in Table 1. In analysis, where there were zero counts for some categories of illness and exposure, confidence intervals (CIs) were estimated using the Cornfield Table 1 Definitions used in the case control study Case Control A diner reporting at least one of diarrhoea or vomiting; or at least two of nausea, abdominal pain or fever, within 72 h of eating at the restaurant between the 5 and 7 of February 2010 A diner not meeting the case definition who ate at the restaurant between the 5 and 7 of February method as implemented in the Stata v10 statistical software package (StataCorp LP, TX, USA). Data did not allow formal multivariate analysis in a logistic regression model. Associations for each exposure identified on univariate analysis were assessed across strata of possible confounding variables where data allowed. Odds ratios for menu options associated with illness were calculated with respective P-values. Microbiological investigation Stool Stool samples were collected from all 16 staff members who were working at the restaurant from 5 to 7 February Symptomatic diners were requested to provide stool samples to their registered general practitioner. All stool samples were cultured for bacterial pathogens at the local hospital microbiology department, with five staff samples and all diner samples tested for norovirus by reverse-transcriptase polymerase chain reaction (RT-PCR) performed at the HPA regional microbiological laboratory in Southampton. Norovirus-positive stool samples were then sent for genotyping at the Centre for Infections (CfI), the national HPA laboratory. Environmental sampling Environmental sampling of the premises was undertaken on 11 February Swabs were taken from food preparation surfaces, and from areas of frequent hand contact in the kitchen areas (e.g. door handles). Swabs were cultured for bacterial pathogens at the HPA regional microbiology laboratory in Southampton. Food The restaurant was supplied with Pacific oysters (Crassostrea gigas) by an oyster company, which sources from beds in Carlingford Lough, situated on the border of Northern Ireland and the Republic of Ireland. A batch of 100 oysters was delivered to the restaurant on 2 February 2010 and served at the restaurant from 5 February until their removal from the menu on 8 February, with 13 portions of six

3 AN OUTBREAK OF NOROVIRUS LINKED TO OYSTER CONSUMPTION 3 oysters being served to 9 separate parties (total of 30 people) between these dates. None of the restaurant staff consumed any of the oysters. At the time of notification, 15 oysters from the batch were available (following wastage of the remainder) and were sent for processing at the Centre for Environment, Fisheries and Aquaculture Science (Cefas) laboratory in Weymouth. Oyster samples were tested for the presence of norovirus using proteinase K digestion of hepatopancreas tissue, 11 followed by RNA extraction using magnetic silica then one-step RT-PCR, as previously described elsewhere. 12 Homogenate from norovirus-positive shellfish and water-only samples were extracted in parallel as positive and negative controls, respectively. Norovirus RNA isolates from the oysters were then forwarded to CfI for genotyping. Results Descriptive epidemiology A total of 30 questionnaires were sent to people who ate at the restaurant between the 5 and the 7 of February as outlined above. Of these, 26 responses were received comprising 11 cases and 15 controls. Key characteristics are outlined in Table 2. The main gastrointestinal symptoms reported were diarrhoea, vomiting and nausea, with a range of onset times Table 2 Key characteristics of diners (cases and controls) Diners Number reporting Cases 11 Controls 15 Age, median (range) 39 (3 75 years) Symptoms Diarrhoea 8 Vomiting 6 Nausea 7 Abdominal pain 4 Fever 1 Headache 3 Lethargy 3 Chill/drop in temperature 3 Tingling sensation 1 Illness onset time after eating (h), Duration of illness (days) from 5 to 50 h after eating at the restaurant. The median duration of illness was 2 days (Table 2). The epidemic curve for this outbreak is displayed in Fig. 1, together with key events of the initial outbreak investigation. In addition to the symptomatic diners, it was discovered that one of the kitchen staff was also unwell, reporting symptoms of diarrhoea and vomiting starting at work after the evening service on 5 February. This unwell member of staff, who works as a pastry chef, did not handle or consume oysters over the preceding week. His symptoms lasted approximately 24 h, during which time he reported the use of a private toilet above the restaurant (separate from kitchen and dining areas). He was excluded from work until 48 h after resolution of symptoms. Analytical epidemiology Table 3 shows the consumption of foods by cases and controls. Starter 7 (oysters, P ¼ ), Main 5 (lamb, P ¼ ) and Dessert 4 (Crème Brulée, P ¼ ) were significantly associated with illness (Table 4). As all of the cases ate oysters, the calculated odds ratio tends towards infinity; the lower limit of the 95% CI for this odds ratio is Similarly the calculated odds ratio for lamb also tends towards infinity; the 95% CI lower limit is 3.8 (Table 4). Within this data set, there is a positive correlation between consumption of oysters and either lamb (P ¼ 0.02) or crème brulée (odds ratio 10, 95% CI: ). Confounding may therefore explain the apparent correlations between one or more exposures and illness. With insufficient data to support a multivariate model, we undertook subset analysis of diner food consumption to Cases Diners Staff Notification to HPA OCM Onset date Fig. 1 Date of onset of symptoms among diners and staff. OCM, outbreak control meeting.

4 4 JOURNAL OF PUBLIC HEALTH Table 3 Food consumed by diners as per questionnaire returns Menu options Cases Controls Ate Not eaten Ate Not eaten Starter Starter Starter Starter Starter Starter Starter 7 (oysters) Shared starter Main Main Main Main Main 5 (lamb) Main Side Side Side Side Side Side Dessert Dessert Dessert Dessert 4 (Crème Brulée) Dessert Dessert Dessert Table 4 Menu options significantly associated with illness Menu option Odds ratio 95% CI P-value Starter 7 (oysters) Main 5 (lamb) Dessert 4 (Crème Brulée) further assess possible confounding. In particular, this addressed subsets of oyster consumption with crème brulée or with lamb consumption. This separate analysis of diner subsets shows that the association of illness with oyster consumption remains highly significant (P, 0.001) in those diners who neither ate lamb nor crème brulée. Furthermore, oyster consumption remains positively associated with illness in those diners who also ate crème brulée, though this association does not reach statistical significance. There was insufficient data to allow analysis of an association between oyster consumption and illness in those who ate lamb. Amongst the diners who ate oysters, there is a very weak though statistically insignificant association between illness and consumption of lamb (P ¼ 0.46) and crème brulée (P ¼ 0.46). Insufficient data prevents analysis of the association between these two exposures and illness among diners who did not eat oysters. Microbiological results Stool All sixteen staff members who were working at the restaurant during the study period provided stool samples, compared with 3 of the 11 symptomatic diners. Unfortunately, one of the diner samples was discarded due to incorrect labelling of the specimen, leaving two diner samples available for testing. All stool samples tested negative for bacterial pathogens. Five staff specimens were tested for norovirus with only the symptomatic chef testing positive (genogroup II). Both diner samples tested positive for norovirus, with genogroup I isolated from one sample and genogroup II from the other. Environmental sampling A visual inspection of the premises was satisfactory. However, environmental swabs taken from two kitchen door handles showed an undesirable level of growth of Staphylococcus aureus. Further swabs from a food preparation surface within the kitchen showed a borderline acceptable aerobic colony count. These results, together with good hygiene practice and training, were discussed by the Environmental Health Officer with the management of the restaurant. Food RNA of norovirus genogroups I and II were isolated from the oysters submitted for testing. Genogroup I RNA was present at a level of,100 copies/g (theoretical limit of detection of assay is 13 copies/g digestive gland). Genogroup II RNA was present at a level of 1736 copies/g, placing this above the 98th centile of all historical samples tested at Cefas. Norovirus genome sequencing Full sequencing of the norovirus genomes isolated in oyster and stool samples was attempted. Analysis showed that there were several different sequences of norovirus RNA present in the oyster and stool samples whilst this

5 AN OUTBREAK OF NOROVIRUS LINKED TO OYSTER CONSUMPTION 5 unfortunately made further detailed genomic analysis very difficult, such a mixture of distinct strains is typically encountered in shellfish-related norovirus outbreaks 13 and also in outbreaks of viral gastroenteritis associated with sewage contamination of food or drinking water. 14 Discussion Main findings of this study In this study, we present a detailed investigation of an outbreak of gastroenteritis at a UK restaurant incorporating both epidemiological and microbiological analysis. Affected diners suffered from gastrointestinal symptoms of h duration, and analysis of stool from symptomatic individuals confirmed norovirus as the causative infective agent. The high levels of norovirus detected in the oyster samples, combined with the strong correlation of illness with oyster consumption and high attack rate amongst those diners who ate oysters, strongly suggests oysters as the vehicle of norovirus transmission. The consumption of lamb and crème brulée was also associated with illness, though further analysis suggests that these foods are likely to be confounding factors in this study. What is already known on this topic The association of gastroenteritis with the consumption of shellfish is well established, particularly with oysters, which tend to be eaten raw. This link was postulated as early as 1816, when an outbreak of typhoid fever in France was linked to consumption of raw oysters from sewagecontaminated waters. 15 Since then, oysters have been proven to be a vehicle of transmission for a plethora of enteric pathogens ranging from bacteria such as Campylobacter and Salmonella to viral pathogens such as hepatitis A virus and norovirus. 15 The risk of such pathogenic contamination can be reduced through depuration, a process whereby oysters are kept in tanks of continually refreshed seawater for several days following harvesting. Depuration reliably and effectively reduces bacterial contamination of oysters; 16 indeed, levels of Escherichia coli and other faecal coliforms form the basis of the statutory regulation of oyster farming. 17 However, standard depuration procedures are ineffective in cleansing oysters of viral contaminants, 18 and several surveys have shown high detectable levels of norovirus in commercially distributed oysters worldwide including Italy, 19 the USA 20 and Georgia, 21 with higher levels detected in the winter months. Despite this evidence, there is no statutory limit on norovirus levels in oyster distribution in the UK in part a reflection of the technical difficulties in norovirus testing in shellfish. 22 Furthermore, there is a paucity of epidemiological studies in this area, making the quantitative correlation of norovirus levels with clinical symptoms uncertain. 23 It is clear that current oyster food safety standards, which rely on bacterial counts, do not adequately control the risk of viral gastroenteritis. Even class A oysters (i.e. highest standard, which includes Carlingford Lough) are associated with significant rates of norovirus infection as highlighted by current national and international epidemiological data, and the very high levels identified in this study. Despite this evidence, there remain several barriers, which prevent the introduction of norovirus testing in the statutory regulation of oyster farming. Firstly, the technical complexities and financial expense of norovirus testing in shellfish are significant; indeed, there are currently only two laboratories in the UK, which can reliably perform the assay (J. A. Lowther, personal communication). Secondly, as the current shellfish assay techniques rely on a RT-PCR approach, it is unclear to what extent the number of viral genome copies detected reflects the number of infectious norovirus virions within the sample. 23 Thirdly, the majority of studies (including this outbreak) have shown oysters to harbour a mix of various different strains of norovirus; 13 the infectivity of each will depend not only on the dose received, but also on strainspecific virulence factors and host pathogen interactions. 24 What this study adds During the course of this investigation, it became apparent that several restaurants across the UK were receiving reports of diners suffering gastrointestinal illness, all with strong links to oyster consumption. On 11 February 2010, an enforcement letter 25 was issued to UK Environmental Health departments and directors of Trading Standards by the Food Standards Agency regarding the possible link of illness with raw oyster consumption, which recommended enhanced depuration procedures. Following this a national outbreak control team was formed, lead by the HPA and the Food Standards Agency, with cooperation from the Food Safety Authority of Ireland. Members of this outbreak control team participated in the national outbreak meetings. Interestingly, several of these outbreaks were linked with consumption of oysters from Carlingford Lough, though other oyster beds in Scotland and England were also implicated. As a result of this national outbreak investigation, there was a voluntary withdrawal of oyster distribution from Carlingford Lough. 26 Oyster distribution from the area was later permitted following enhanced treatment (consisting of relaying oysters in sewage-free areas and extended warm-water depuration), which was shown to reduce norovirus levels to those detected prior to the outbreak. 27 The increased incidence of oyster-related illness observed nationally within the UK also seems to be reflected

6 6 JOURNAL OF PUBLIC HEALTH internationally within Europe. The European Centre for Disease Prevention and Control has recently reported a total of 65 clusters of norovirus infection linked to oyster consumption, involving a total of 334 cases from January to March 2010 across five European countries: UK, Norway, France, Sweden and Denmark. 28 Limitations of this study The results of this study strongly support oysters as the vehicle of norovirus transmission. However, a causal role for these other food items cannot be completely excluded given the small sample size and restriction of the study to parties where at least one diner consumed oysters. Increasing the number of controls in the study would likely have lead to clearer epidemiological results; in practice, this was not possible as the restaurant management did not release contact details of further diners. In addition to the diners, a pastry chef working at the restaurant also developed norovirus infection, though he exclusively prepared desserts and did not handle or consume any of the oysters. The chef s stool sample confirmed genogroup II norovirus infection, and hence cannot account for transmission of infection to the diner with genogroup I infection. Furthermore, the diner who did become infected with genogroup II norovirus did not eat any of the desserts, making transmission of infection from the pastry chef less likely. Given the contemporaneous high background rate of community norovirus infection, it is probable that the chef was a coincidental case rather than a causal factor in the outbreak. In conclusion, the evidence that oysters are a vehicle for norovirus transmission is now irrefutable, and consumption of raw oysters is associated with a risk of illness, especially in the winter months. However, whilst norovirus contamination of oysters poses a potential risk to human health, there is significant uncertainty surrounding the quantitative correlation between oyster norovirus levels and consumer illness. Continued research in this area should help to further our understanding of this crucial correlation, as well as identify ways in which viral depuration can be enhanced. Until then, the catering industry should be aware and the public should continue to be informed of the risk of viral gastroenteritis posed by raw oyster consumption. References 1 Patel MM, Hall AJ, Vinjé Jet al. Noroviruses: a comprehensive review. J Clin Virol 2009;44(1): Health Protection Agency. Norovirus laboratory reports of all identifications by region reported to the Health Protection Agency, Centre for Infections, England and Wales, hpa.org.uk/webw/hpaweb&hpawebstandard/hpaweb_c/ ?p= Hawker J, Begg N, Blair I et al. Communicable Disease Control Handbook, 2nd edn. 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7 AN OUTBREAK OF NOROVIRUS LINKED TO OYSTER CONSUMPTION 7 20 DePaola A, Jones JL, Woods J et al. Bacterial and viral pathogens in live Oysters: U.S. market survey Appl Environ Microbiol 2010;76(9): Gentry J, Vinjé J, Guadagnoli D et al. Norovirus distribution within an estuarine environment. Appl Environ Microbiol 2009;75(17): Schultz AC. Comparison of methods for detection of norovirus in oysters. Int J Food Microbiol 2007;114(3): Lowther JA, Avant JM, Gizynski K et al. Comparison between quantitative real-time reverse transcription PCR results for norovirus in Oysters and self-reported gastroenteric illness in restaurant customers. J Food Prot 2010;73(2): Donaldson EF, Lindesmith LC, Lobue AD et al. Viral shapeshifting: norovirus evasion of the human immune system. Nat Rev Microbiol 2010;8(3): Houston C. Possible illness associated with the consumption of raw oysters. Food Standards Agency correspondence: reference ENF/ E/10/ enf/e/10 (11 February 2010). 26 Food Safety Authority of Ireland. Withdrawal of raw oysters from Carlingford Lough. Alert notification news_centre/food_alerts/raw_oysters_10.html (15 February 2010). 27 Doré B, Keaveney S, Flannery J et al. Management of health risks associated with oysters harvested from a norovirus contaminated area, Ireland, February March Euro Surveill 2010;15(19): pii: Westrell T, Dusch V, Ethelberg S et al. Norovirus outbreaks linked to oyster consumption in the United Kingdom, Norway, France, Sweden and Denmark, Euro Surveill 2010;15(12): pii:19524