Eurosynthèse
Volume 07 N°04 - April 2002

B. Lopman1, Y. van Duynhoven2, F. X. Hanon3, M. Reacher1, M. Koopmans2 and D. Brown1 on behalf of the consortium on Foodborne Viruses in Europe*

1 Public Health Laboratory Service, Colindale, London, England.
2 Research Laboratory for Infectious Diseases, National Institute for Public Health and the Environment (RIVM), The Netherlands
3 Department of Virology and Epidemiology Research, Statens Serum Institute, Department of Virology, Copenhagen, Denmark.

This report describes a survey of national laboratory capabilities of diagnostics and surveillance databases for foodborne viruses among the "Foodborne Viruses in Europe" consortium.

All the countries have laboratories that can test for HAV antibody in human serum. Eight of the ten surveyed European countries maintain a national database of HAV cases. Food can be tested for the presence of HAV in Finland, Italy, Spain, France and Denmark.

All surveyed countries have at least one laboratory that tests for Norwalk-like virus (NLV) by reverse transcriptase-polymerase chain reaction and all also have the capability to use electron microscopy. Five countries maintain a national database of NLV cases and nine maintain a national database of NLV outbreaks. Almost all participant countries have laboratories that can test for NLV in food items including shellfish.

The importance of food and water in the transmission of Norwalk-like viruses (NLVs) and Hepatitis A virus (HAV) is increasingly recognised. Outbreaks spread by these modes have the potential to involve large numbers of people, be widely geographically spread and, perhaps, introduce new variants to an area. Virus typing, using modern molecular methods, has shown how contaminated food from one country has resulted in outbreaks in another country from imported food. The risk of hepatitis A (HAV) outbreaks is substantial due to waning population immunity as a consequence of safe water and sanitation in Europe. Norwalk-like viruses (NLVs) cannot be cultured in the laboratory, thus, diagnosis is based on electron microscopy (EM) or, increasingly, on molecular techniques. HAV is diagnosed routinely by the detection of virus-specific antibodies.

There are numerous reports of foodborne outbreaks of NLV and HAV, but the true incidence of these diseases and the contribution of these outbreaks to the disease burden remain unclear.

The "Foodborne viruses in Europe" research project was recently funded by the European Union to obtain better information on the impact of foodborne viruses within the EU. Groups were included in the network based on their interest and track record in viral gastro-enteritis. The participant countries have networked their virological and epidemiological surveillance in order to detect transna-

tional outbreaks as well as elucidate transmission routes. In order to facilitate the design of a European database, we surveyed all the participant countries of the "Foodborne Viruses in Europe" project about their laboratory capabilities as well as the databases that record cases and outbreaks of viral gastro-enteritis.

Methods

Table

Databases and laboratory capability for foodborne viruses among the consortium for Foodborne Viruses in Europe

VHA / HAV= Virus de l’hépatite A / Hepatitis A virus

VDN / NLV= Virus de Norwalk / Norwalk-like virus

EIA = enzyme immuno assay;

EM=microscopie électronique / electron microscopy;

RT-PCR= reverse transcriptase polymerase chain reaction

* Surveillance pilote / Pilot surveillance

A questionnaire was sent by email to twelve participant institutions (from ten countries) in the "Foodborne viruses in Europe" project (see participants list) as well as the Institute of Microbiology and Immunology (Slovenia), which was not officially a member of the group. Groups were included in the project based on their interest in NLV and HAV molecular biology, epidemiology and/or food safety. Information was collected on 1) the diagnostic techniques used in routine laboratories 2) the number and types of databases used that store information on NLV and HAV infection ; and 3) the capacity for environmental testing for these viruses.

A database was defined as: any organised set of electronic or paper-based information on individual human cases or summaries

of outbreaks. A national database was considered one designed to collect information from all geographic regions of a country, but no stipulations were made concerning the overall or regional levels of reporting.

Results

A completed survey questionnaire was returned from all 10 countries.

Hepatitis A

Human diagnostics

All countries have the capability to test for HAV immunoglobulin (Ig) in human serum but, the number of laboratories performing tests in each country varies widely (table). Laboratories in Germany, Spain, France, The Netherlands and Finland test for HAV RNA by reverse-transcription polymerase chain reaction (RT-PCR).

Surveillance

All countries except France and Sweden have a national database of HAV cases (though a pilot surveillance has begun in France) (figure A). Of those countries that do maintain HAV case databases, all contain information from laboratory reports except for Germany. England and Wales, Italy, Finland, and the Netherlands maintain additional databases that receive reports from primary care doctors and other health care institutions. In addition, HAV databases derived from a range of special studies also exist: an outbreak survey of public health physicians in 1998 (England and Wales), a pilot surveillance of HAV infection (France), and a survey on travellers before vaccination (Slovenia).

Environmental testing

Food can be tested for the presence of HAV genetic material by RT-PCR by laboratories in Finland, Italy, Spain, France, and Denmark. Laboratories in these five countries as well as England and Wales can test for HAV in water and drinks.

Norwalk-like virus

Human diagnostic capability

All countries use RT-PCR and EM to detect NLVs, though in some countries EM is now used as a last line of detection (Table). Two

laboratories in England and Wales use an enzyme immuno assay (EIA) based on recombinant NLV.

Surveillance

All countries, except Spain, maintain at least one (laboratory-based) database of cases of NLV. In the Netherlands databases also hold information from primary care and other healthcare providers. In addition, there are historical databases from surveys such as the Dutch NIVEL case-control study, and SENSOR cohort study as well as a study of outbreaks in the western regions of France. Also in France, historical databases exist from a study of children (1995-98). England and Wales, Germany, Finland, Slovenia and Denmark have national databases of NLV cases (figure B).

Databases of NLV outbreaks are kept in every country. These databases are all laboratory-based apart from one based on primary care data (France), and two others based on health care facility data in Denmark and The Netherlands. There are also databases from special studies of structured outbreak surveillance in England, and Wales and The Netherlands. Except for Italy, outbreak databases in every country are designed to collect national data (figure C), ‰ ‰ although this does not necessarily mean that all outbreaks that are investigated are then reported. In France and Denmark, only food- or waterborne outbreaks were reported.

Environmental testing

Laboratories that can test human stools also have the ability to test food animal stools, although only Finland, England and Wales, France, Denmark, and The Netherlands perform such tests. Most countries also have laboratories that test foods (namely shellfish) as well as water and drinks (see table).

Discussion

This inventory of laboratory capacity is the first step towards harmonisation of laboratory and surveillance of foodborne viruses in participating countries. Both the capability and the infrastructure of laboratories testing for foodborne viral pathogens in Europe vary widely. The sharing of samples and experience through the Foodborne Viruses in Europe consortium will however make these diverse capabilities complementary. Some countries have a more centralised diagnostic service with nearly all testing of foodborne viruses concentrated in one or two laboratories. In other countries, such as England and Wales, Germany, The Netherlands, and Italy, diagnostics are provided in many laboratories.

Although the majority of countries in this survey have laboratories that can test for HAV infection in humans, many do not test for HAV in food, water or drinks and therefore, food and waterborne infections cannot be microbiologically proven without international assistance. All countries can test human faeces for NLV by RT-PCR and EM. In comparison to EM, the RT-PCR is a substantially more sensitive diagnostic tool, able to detect virus up to two weeks after infection . Thus, the use of RT-PCR could increase the laboratory ascertainment of cases and outbreaks of NLV. Due to the genetic diversity of NLVs it has been difficult to develop a sufficiently sensitive and specific assay. In order to harmonise diagnostic methods used across the network, laboratories have tested a representative panel of stool samples using the different assays used among the network participants. The results of this evaluation will be presented elsewhere.

Testing of shellfish can be performed in most countries though satisfactory methods have not been standardised. There are no established tests for assaying other foods, and one of the aims of the "Foodborne Viruses in Europe" is to develop such techniques. Laboratories in Finland, The Netherlands, England and Wales, Denmark, and France have used their facilities to test food animals for NLV, thus capturing the possibility of zoonotic infection. Though transmission of NLV from animal to humans has not been demonstrated, evidence of the potential has been mounting in recent years. NLV genes have been detected in pigs and cattle, and the genetic material of bovine caliciviruses were shown to be very similar to human NLV.

All countries have at least one database of NLV outbreaks and all but Italy have a database with national coverage. Ascertainment of NLV gastroenteritis by routine surveillance has been shown to be poor since the condition is typically mild in that it does not cause the affected individuals to seek medical attention. Though the level of ascertainment may vary widely, we believe that an international database that captures outbreaks would be most appropriate for the "Foodborne Viruses in Europe" network.

Diagnostics and, therefore, surveillance of foodborne viruses are rapidly evolving fields. Molecular techniques used to detect viral RNA (by RT-PCR assay) and virus particles (EIA) are increasingly used in diagnostic laboratories. And, many of the national databases and surveillance networks that are referred to in these reports have been in existence for a short period of time. For example, reporting of NLV cases in Germany to a central database began as recently as January 2001.

Through the use of molecular typing, a transnational foodborne outbreak of NLV where contaminated raspberries from Slovenia caused infections in Europe and Canada has been described. Large scale food- and waterborne outbreaks have been documented a number of times but this proof of a transnational outbreak demonstrates the potential health impacts of the diffuse distribution of modern foodstuff industry. Through the creation of an international epidemiological and molecular database, the "Foodborne Viruses in Europe" project will study how common such outbreaks are and which are the transmission routes associated with most outbreaks. It will also enable the detection of epidemics and of epidemic variant viruses at an early stage.

* Participants

Pays-Bas / The Netherlands: Dr. M. Koopmans, Dr. H. Vennena, Dr. Y. van Duynhoven, Dr. W. van der Poel, National Institute of Public Health and the Environment; Bilthoven; Finlande / Finland: Dr. K-H von Bonsdorff, L. Maunula, Helsinki University; Danemark / Denmark: Dr. B. Böttiger, Dr. K. Mölbak, F.X. Hanon, Statens Serum Institute, Copenhagen; Suède / Sweden: Dr. L. Svensson, Dr. K-O Hedlund, Swedish Institute for Infectious Disease Control, Solna; RU / UK: Dr. D. Brown, Dr. M. Reacher, Dr. J.Green, B. Lopman, Public Health Laboratory Service, London; Allemagne / Germany: Dr. E. Schreier, Dr. H. Gelderblom, Robert Koch Institute Berlin; Espagne / Spain: Dr. A. Sanchez, Instituto de Salud Carlos III, Madrid; Dr. A. Bosch, Universitat de Barcelona, Barcelona; Dr. J. Buesa, Universitat de Valencia; France: Dr. F. LeGuyader, IFREMER, Nantes; Dr. P. Pothier, Dr. E. Kohli, Laboratoire de Virologie, Centre Hopitalier Universitaire Dijon; Italy / Italy: Dr. F. Ruggeri, Dr. D. DeMedici, Instituo di Superiore di Sanitá, Rome; Slovénie / Slovenia: Dr. M. Poljsak-Prijatelj, Institute of Microbiology and immunology, Ljubljana.