Background
West Nile (WN) virus is a mosquito-transmitted flavivirus which belongs to the Japanese encephalitis virus group. It occurs throughout Africa, the Middle East, southern Europe, Russia, India and Indonesia, and was recently introduced into North America [
1‐
3]. Migratory birds are involved in the transmission cycle of this virus as amplifying hosts, and humans and horses are considered to be accidental dead-end hosts [
1,
4]. In humans, the majority of WN virus infections cause a non-symptomatic or a mild flu-like illness. However, some infections can cause encephalitis which may lead to death, particularly in elderly patients [
3]. WN virus is a clear example of the tremendous impact that virus spread and evolution can have on human beings. From 1999 to 2006 there were 8422 neuroinvasive WN cases (including 889 fatalities) reported in the United States [
5]. The incidence of WN virus in Europe is comparatively poorly studied and the risk for a similar epidemic, although low, cannot be precisely estimated [
6]. The availability of reliable serological assays such as the enzyme immunoassay (EIA), immunofluorescence assay (IFA) or neutralisation test (NT) is an important prerequisite for the clinical diagnosis and epidemiological surveillance of WN virus infections.
A major problem for WN serological assays is their high degree of cross-reactivity with antibodies produced in response to other simultaneous and/or previous flavivirus infections [
7]. False positive results are due to the cross-reactivity of antibodies specific for related epitopes found on other flaviviruses (e.g. Saint Louis encephalitis-, dengue-, yellow fever-, tick-borne encephalitis-, or Japanese encephalitis virus) induced by natural infection or vaccination. This is mainly true for IgG- and in some cases for IgM-antibodies. Since differentiation of a specific immune response is difficult, a fourfold increase in antibody titre in follow-up patient sera is mandatory for a positive diagnosis [
8].
Several research laboratories have developed serological assays for WN virus infection and a number of commercial test kits are now available [
9]. However, the performance of these methods varies considerably between laboratories. Comprehensive external quality control studies for WN serology have not yet been performed and little information is available about the relative and overall proficiency in different laboratories. Comparative testing of well-characterised samples is the best method of identifying weaknesses of single laboratories or of certain methodological components. The aim of this study was to assess the diagnostic accuracy across participating laboratories and the tests they use by performing the first international external quality assurance (EQA) study for the serological detection of WN virus infection.
Methods
Participants and recruitment
Twenty-seven laboratories from 20 different countries participated in this EQA programme, including 20 laboratories from Europe, three from the Middle East, three from North or South America and one from Africa. A complete list of participants is given in the acknowledgements section. The study was announced as an EQA study on diagnostic proficiency run by the European Network for diagnostics of 'Imported' Viral Diseases (ENIVD), including publication of the results in a comparative and anonymous manner. Participation was open and free of charge to all laboratories performing WN diagnostics. Selection of invitees was based on the register of ENIVD members as well as on their contributions to the literature relevant to this topic.
Preparation of test samples
Test samples for the proficiency panel were generated by diluting well-characterised human sera with fresh-frozen plasma tested and confirmed to be negative for HIV, hepatitis B-, hepatitis C-, WN- and non-WN-flaviviruses. After dilution, the enriched serum samples were heat-treated (56°C, 1 h), frozen and lyophilised in aliquots of 100 μl to prepare proficiency panels consisting of 10 test samples. As positive controls the panel comprised aliquots of four antisera positive for WN antibodies purchased from SeraCare Life Sciences, Milford, MA, USA. For specificity controls, aliquots of three antisera containing antibodies reactive with heterologous flaviviruses (tick-borne encephalitis-, yellow fever- and Dengue virus) provided by reference laboratories of our network and one serum with a known unspecific reactivity against cell and mitochondrial structures provided by EUROIMMUN AG, Lübeck, Germany were included. Two additional aliquots from confirmed seronegative samples served as negative controls. Two sets of this EQA panel were tested for specific activity by two expert laboratories to confirm the quality of the samples after preparation.
Distribution of test samples and given instructions
All samples were sent out by regular mail and arrived within 1 week after sending, according to the date of arrival provided by the participants. It was recommended that the samples be resuspended with 100 μl distilled water and centrifuged for 5 min to remove any aggregates before testing. The participants were asked to analyse the material by the diagnostic methods they routinely used for the serological detection of WN virus infection. There was no obligation concerning the test procedure to be used but information concerning the type/format (EIA, IFA, NT) and whether it was an in-house assay or a commercial kit was requested. No further information regarding the detailed processing of the NTs (format, cell line used, 50% or 90% calculation, reference etc.) were provided by the participating laboratories.
Evaluation of participants' results
The following two criteria were selected as the minimum requirements for successful overall proficiency, scored with 10 points (= 100%). First, laboratories had to detect the four WN-positive samples irrespective of the differentiation between IgM and IgG; this means that at least one of these tests had to give a positive result. Second, the antisera containing cross-reactive antibodies to heterologous flaviviruses (tick-borne encephalitis-, yellow fever- and dengue virus) or the known unspecific serum should not give a positive result and/or should be recognised as being unspecific. Equivocal or borderline results with the non-WN-flavivirus positive samples were treated as negative. The indeterminate result of Lab. No. 15 in one of the negative samples was identified as such and was not used in the evaluation. False positive or negative results as well as non-clarified results with clinical consequences were given a score of -1. The differentiation between IgM and/or IgG results was considered separately and gave additional information concerning the quality of the laboratory diagnostics. Data collected were entered into Microsoft Excel (Microsoft Corp., Bellingham, WA, USA) and analysed using SPSS 14.0 for Windows. Results with respect to categorised variables were analysed by the chi-square test. Whether or not common technical factors or particular samples influenced the performance of the participating laboratories was assessed by univariate and multivariate logistic regression. A p-value < 0.05 was considered to indicate statistical significance.
Acknowledgements
The EQA was performed by the European Network for diagnostics of 'Imported' Viral Diseases (ENIVD) presently funded in part by the EC DG SANCO under the programme AIDS and other communicable diseases grant No. SI2.299717 (2000CVG4-26). The authors thank Dr. Stephen Norley, Dr. Regina Schädler and Marcel Müller, BSc for critical reading of the manuscript.
The following 27 laboratories participated in the study:
Europe/Middle East: Centre for Emergency Preparedness & Response, Porton Down, UK; Institut für klinische Mikrobiologie, St. Gallen, Switzerland; Centre for Ecology and Hydrology, Oxford, UK; Rijksinstituut voor Volksgezondheid (RIVM), Bilthoven, The Netherlands; University College Dublin, Dublin, Ireland; Istituto Nazionale per le Malattie Infettive, Rome, Italy; Institute of Tropical Medicine, Antwerpen, Belgium; Institut de Médecine Tropicale du Service de Santé des Armées (IMTSSA), Marseille Armées, France; EUROIMMUN AG, Lübeck, Germany; Medizinische Universität Wien, Vienna, Austria; UBIVE Institut Pasteur, Lyon, France; Instituto Nacional de Saúde Dr. Ricardo Jorge (INSA), Águas de Moura, Portugal; Bernhard Nocht Institut, Hamburg, Germany; National Centre for Microbiology, Majadahonda (Madrid), Spain ; National Centre for Epidemiology, Budapest, Hungary; Erasmus University Rotterdam, Rotterdam, The Netherlands; University of Ljubljana, Ljubljana, Slovenia; Paul-Ehrlich-Institut, Langen, Germany; Laboratorio Central de Veterinaria, Algete (Madrid), Spain; Cantacuzino Institute, Bucharest, Romania; Kimron Veterinary Institute, Beit Dagan, Israel; Pasteur Institute of Iran, Teheran, Iran; Sheba Medical Center, Ramat-Gan, Israel; The Americas: Laboratorio Nacional de Salud Guatemala, Villa Nueva, Guatemala; Instituto de Diagnostico y Referencia Epidemiologicos (InDRE), Mexico City, Mexico; Focus Technologies, Inc., Cypress, USA; Africa: Special Pathogens Unit, National Institute for Communicable Diseases, Johannesburg, South Africa.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MN designed the study together with HZ and was responsible for data acquisition. MN and ODM were responsible for evaluation and interpretation of data and wrote the manuscript. DA provided statistical analysis. HZ provided review and expert advice. All authors read and approved the final manuscript.