Review
Review: Norovirus prevalence in Belgian, Canadian and French fresh produce: A threat to human health?

https://doi.org/10.1016/j.ijfoodmicro.2011.09.013Get rights and content

Abstract

Foodborne viruses, especially noroviruses (NoV), are increasingly reported as the cause of foodborne outbreaks. NoV outbreaks have been reported linked to fresh soft red fruits and leafy greens. Belgium, Canada and France were the first countries to provide data about the prevalence of NoV on fresh produce. In total, 867 samples of leafy greens, 180 samples of fresh soft red fruits and 57 samples of other types of fresh produce (tomatoes, cucumber and fruit salads) were analyzed. Firstly, the NoV detection methodology, including virus and RNA extraction, real-time RT-PCR and quality controls were compared among the three countries. In addition, confirmation and genotyping of the NoV strains was attempted for a subset of NoV positive samples using conventional RT-PCR targeting an alternative region followed by sequencing. Analysis of the process control showed that 653, 179 and 18 samples of the leafy greens, soft red fruits and other fresh produce types were valid for analysis based on the recovery of the process control. NoV was detected by real-time RT-PCR in 28.2% (N = 641), 33.3% (N = 6) and 50% (N = 6) of leafy greens tested in Canada, Belgium and France, respectively. Soft red fruits were found positive by real-time RT-PCR in 34.5% (N = 29) and 6.7% (N = 150) of the samples tested in Belgium and France, respectively. 55.5% (N = 18) of the other fresh produce types, analyzed in Belgium, were found NoV positive by real-time RT-PCR. Conventional RT-PCR resulted in an amplicon of the expected size in 19.5% (52/266) of the NoV positive samples where this assay was attempted. Subsequent sequencing was only successful in 34.6% (18/52) of the suspected amplicons obtained by conventional RT-PCR. From this study, using the described methodology, NoV genomes were frequently detected in fresh produce however sequence confirmation was not successful for the majority of the samples tested. Infection or outbreaks were rarely or not known to be related to the NoV positive samples. With the increase in sensitivity of the detection methodology, there is an increasing concern about the interpretation of positive NoV results by real-time amplification. Strategies to confirm the results by real-time RT-PCR should be developed in analogy with the detection of microbial pathogens in foods. Detection might indicate contact with NoV in the fresh produce chain. Consequently, a potential risk for infection cannot be excluded but the actual risk from RT-PCR NoV positive produce is still unknown. Studies should be designed determining the probability of infection related to the presence or levels of NoV genomic copies.

Highlights

► Investigation of fresh produce in Belgium, Canada and France for norovirus by real-time RT-PCR. ► Norovirus detection on the majority of fresh produce samples by real-time RT-PCR could not be confirmed. ► The risk to human health from RT-PCR norovirus positive fresh produce is unknown. ► Strategies to confirm the results by real-time RT-PCR should be developed. ► Studies should be designed determining the probability of infection related to the presence or levels of norovirus genomic copies.

Introduction

Foodborne viruses, mainly caliciviruses (noroviruses), were the second most reported cause of foodborne outbreaks in 2007–2008 in the EU and the most common cause of foodborne outbreaks in the US in 2007 (Centers for Disease Control and Prevention, 2010, European Food Safety Agency, 2010). Implicated foodstuffs were shellfish, buffet meals, and fruits, although almost 50% were not specified or unknown. A review of food- and waterborne outbreak events due to norovirus (NoV) between 2000 and 2007 revealed that in 42.5% of the cases the food handler was responsible for the outbreak, contaminating sandwiches and catered meals, followed by water (27.5%), bivalve shellfish (17.5%) and fresh produce (12.5%) (Baert et al., 2009). The prevalence of NoV is well documented in shellfish (Boxman et al., 2006, Croci et al., 2007, Nishida et al., 2003, Terio et al., 2010) and surface water (Lodder and de Roda Husman, 2005, Pusch et al., 2005, Victoria et al., 2010). Until recently, no data was available about the prevalence of NoV on fresh produce. Nevertheless, NoV outbreaks have been reported linked to leafy greens (Ethelberg et al., 2010, Gallimore et al., 2005, Makary et al., 2009, Wadl et al., 2010). Soft red fruits have also been implicated in NoV outbreaks (Le Guyader et al., 2004, Maunula et al., 2009). Rapid alerts related to the detection of NoV in lettuce and soft red fruits have been noted (RASSF, 2010). The lack of appropriate methods to detect NoV on fresh produce has hampered testing of this food commodity–pathogen combination. Over the last decade, several methods for virus, and in particular NoV extraction and detection, have been evaluated (Baert et al., 2008b, Butot et al., 2007). In Europe, the CEN/TC275/WG6/TAG4 working group is harmonizing detection methods for NoV and hepatitis A virus (HAV) in foods with the aim to recommend a standardized detection method. In Canada, standard methods are published online in Health Canada's Compendium of Analytical Methods (http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume5/index-eng.php).

NoV is introduced on fresh produce by contact with contaminated fecal material, which can occur at any point during food production, harvest, processing or preparation. At the pre-harvest level, contact with polluted irrigation water or organic fertilizer is possible (Wei et al., 2010). During harvest, in particular during handpicking of crops, an infected person could transmit the virus and at the post-harvest level the produce could be contaminated by contact with polluted process water or during food preparation by infected food handlers (Baert et al., 2008a). Fresh produce is on the one hand promoted as part of a healthy diet, resulting in an increased consumption, but on the other hand could pose a public health risk as it is eaten raw.

To investigate the prevalence and contamination level of NoV on fresh produce, survey data were collected in Belgium, Canada and France. In this review, published and unpublished Belgian, Canadian and French data regarding the detection of NoV on fresh produce are summarized. This presentation allows for evaluation of the detection methodology, including the quality controls, used by the three countries. An elution–precipitation approach was used in Belgium and France while an adsorption–elution–ultrafiltration approach was conducted in Canada. The data provide an overview of NoV detection in different categories of fresh produce: leafy greens, soft red fruits and other (consisting of tomato samples, cucumber slices and fruit salads). Each jurisdiction encountered difficulties associated with confirmation of positive real-time RT-PCR results by conventional RT-PCR and sequencing. The critical question addressed is how to interpret NoV positive results on fresh produce by real-time RT-PCR amplification with regard to the risk for human health.

Section snippets

Origin of fresh produce

In total 93 Belgian fresh produce samples were analyzed for the presence of NoV. A screening study performed during April–May 2009 investigated 75 fruit products derived from Belgian fresh produce processing companies: 10 samples of raspberries (originating from Poland/Serbia), 15 samples of fruit salads (mixed fruit cut and prepared in Belgium), 30 samples of cherry tomatoes and 20 samples of strawberries (originating from Spain) (Stals et al., 2011). Another 18 samples, including leafy greens

Evaluation of the NoV detection methodology on fresh produce

The detection strategy for NoV is presented in Fig. 2. The detection method is a multistep approach consisting of two major parts; (i) the viral extraction procedure and (ii) the real-time RT-PCR assay. Consequently, the inclusion of quality controls in both stages is mandatory in order to correctly interpret test results. To control the viral extraction procedure, a process control is a prerequisite, i.e. a virus added to the sample portion at the earliest opportunity prior the virus

Conclusions and perspectives

The detection methodology for NoV, consisting of the virus and RNA extraction procedure followed by real-time RT-PCR has improved in sensitivity the last years. A detection strategy using appropriate analytical controls is a prerequisite to detect NoV in foods. A process control is required to judge if test results are valid and if they can be further analyzed for the presence of NoV. Furthermore, a reverse transcription control is a prerequisite to examine whether inhibition of the molecular

Acknowledgements

This work was supported by a postdoc grant (L. Baert) from the Research Foundation–Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen) and by the C-EnterNet program of the Public Health Agency of Canada.

References (62)

  • J.A. Lowther et al.

    Comparison between quantitative Real-Time Reverse Transcription PCR results for Norovirus in oysters and self-reported gastroenteric illness in restaurant customers

    Journal of Food Protection

    (2010)
  • K. Mattison et al.

    The feline calicivirus as a sample process control for the detection of food and waterborne RNA viruses

    International Journal of Food Microbiology

    (2009)
  • D.G. Newell et al.

    Food-borne diseases—the challenges of 20 years ago still persist while new ones continue to emerge

    International Journal of Food Microbiology

    (2010)
  • A. Stals et al.

    Laboratory efforts to eliminate contamination problems in the real-time RT-PCR detection of noroviruses

    Journal of Microbiological Methods

    (2009)
  • A. Stals et al.

    Screening of fruit products for norovirus and the difficulty of interpreting positive PCR results

    Journal of Food Protection

    (2011)
  • V. Terio et al.

    Norovirus in retail shellfish

    Food Microbiology

    (2010)
  • K. Uhrbrand et al.

    Evaluation of a rapid method for recovery of norovirus and hepatitis A virus from oysters and blue mussels

    Journal of Virological Methods

    (2010)
  • H. van den Berg et al.

    Genetic diversity of noroviruses in raw and treated sewage water

    Research in Microbiology

    (2005)
  • L. Baert et al.

    Survival and transfer of murine norovirus 1, a surrogate for human noroviruses, during the production process of deep-frozen onions and spinach

    Journal of Food Protection

    (2008)
  • L. Baert et al.

    Detection of murine norovirus 1 by using plaque assay, transfection assay, and real-time reverse transcription-PCR before and after heat treatment

    Applied and Environmental Microbiology

    (2008)
  • L. Baert et al.

    Reported foodborne outbreaks due to noroviruses in Belgium during 2007: the link between food and patient investigations in an international context

    Epidemiology and Infection

    (2009)
  • A. Bosch et al.

    Analytical methods for virus detection in water and food

    Food and Environmental Virology

    (2011)
  • S. Butot et al.

    Procedure for rapid concentration and detection of enteric viruses from berries and vegetables

    Applied and Environmental Microbiology

    (2007)
  • Centers for Disease Control and Prevention

    Surveillance for foodborne disease outbreaks—United States, 2007

    Morbidity and Mortality Weekly Report

    (2010)
  • M.I. Costafreda et al.

    Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples

    Applied and Environmental Microbiology

    (2006)
  • B. Cotterelle et al.

    Outbreak of norovirus infection associated with the consumption of frozen raspberries, France, March 2005

    Eurosurveillance

    (2005)
  • M. D'Agostino et al.

    Nucleic acid amplification-based methods for detection of enteric viruses: definitions of controls and interpretation of results

    Food and Environmental Virology

    (2011)
  • A.K. da Silva et al.

    Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: Different behaviors of genogroups I and II

    Applied and Environmental Microbiology

    (2007)
  • B. Dore et al.

    Management of health risks associated with oysters harvested from a norovirus contaminated area, Ireland, February–March 2010

    Eurosurveillance

    (2010)
  • European Food Safety Agency

    The community summary. Trends and sources of zoonoses and zoonotic agents and foodborne outbreaks in the European Union in 2008

    The EFSA Journal

    (2010)
  • S. Ethelberg et al.

    Outbreaks of gastroenteritis linked to lettuce, Denmark, January 2010

    Eurosurveillance

    (2010)
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