Introduction
In addition to lifestyle changes, migration and other factors, travel is one of the most potent reasons for the emergence of infections, and the current volume (e.g., 1600 million air travellers per year), speed and distance of travel are unprecedented [1]. Numerous viral outbreaks in recent years, such as Ebola haemorrhagic fever in Democratic Republic of Congo (1995), Uganda (2000) and Gabon and Republic of Congo (2001-2003), West Nile fever in the United States (US) in 1999 and severe acute respiratory syndrome (SARS) in China in 2002 [2-6], serve as reminders that severe infections can be imported into Europe by travellers coming from regions with a high incidence and then spread quickly. There have been several reports in the last ten years from various European countries regarding the import of suspected or confirmed cases of viral haemorrhagic fever (VHF) and SARS which support these concerns [TABLE 1].

Without appropriate diagnostic tests, there is a risk that such infections will be diagnosed incorrectly because clinicians are often unfamiliar with the symptoms. Furthermore, examination of the patient's close personal contacts (including hospital staff) will often be unsuccessful and will carry the additional risk of disease transmission.

Since the anthrax incidents in the US in 2001 and the worldwide terror attacks in recent years we have all become aware of the need in every country (including those of Europe) to be prepared for dealing with people who have been exposed to or infected with agents of bioterrorism (BT). This includes the rapid detection and surveillance of putative agents and prompt response and communication [23-27]. The viral agents that are more likely to be used for bioterrorist attack are not commonly encountered in the majority of clinical microbiology laboratories [TABLE 2], and with the exception of smallpox virus most of these agents are occasionally isolated from patients who have been naturally infected.

The early recognition of a bioterrorist event therefore depends on trained medical and laboratory personnel (especially at the community healthcare level), on specific and sensitive laboratory techniques allowing the early identification of potential bioterrorist agents and on a closer cooperation between global organisations, such as the World Health Organization (WHO), and entities such as the European Union (EU) and individual countries. It is impossible to know in advance which newly emergent pathogens might be used by terrorists, and it is therefore imperative that efforts for BT preparedness be coordinated with disease surveillance and outbreak response activities.

Having appropriate detection strategies in support of surveillance and control of imported, rare and emerging viral infections are dependent upon having established specific and sensitive laboratory diagnostic tests. The European Network for Diagnostics of “Imported” Viral Diseases (ENIVD), established in 1998 in response to decision no. 2119/98/EC [28], fulfils important tasks in this field of research [29]. These tasks include (i) providing mutual help by the exchange of diagnostic samples, reagents, methodologies and expertise; (ii) improving the performance of diagnostic tests by running external quality assurance (EQA) programmes; and (iii) organising and coordinating international cooperations with the European ’Surveillance network group’, and other national and international organisations such as the Centers for Disease Control and Prevention (CDC) in the US, the WHO and the Pan American Health Organization (PAHO). Presently, the network comprises 44 expert laboratories spanning 21 EU member states and 4 non-EU countries as permanent members. Here, we present the results and conclusions of our EQA activities carried out in the last five years in which several expert laboratories, from both inside and outside Europe, were invited to participate with the aim of evaluating and improving their laboratory techniques.

Methods
Between 1999 and 2004 several EQA programmes have been established to assess the quality of serological and/or molecular diagnostics of hantavirus, dengue virus, filovirus, Lassa virus, orthopox virus and SARS-CoV infection [TABLE 3]. A total of 93 invited expert laboratories from 41 European and non-European countries participated at least in one of these studies. The selection of invitees was based on the register of ENIVD members as well as on their literature contributions relevant to each of the topics (for the detailed lists of participants see references in table 3). In the case of the SARS-relevant EQA studies, invitees were members of the international WHO SARS Reference and Verification Laboratory Network or of national and regional SARS reference laboratories. Each of the studies was announced as an EQA study of diagnostic proficiency, which included the certification and publishing of the results in a comparative and anonymous manner.

For the EQA of serological diagnostics each participant received a coded panel of 15 or 20 freeze-dried human sera that also included negative controls. The positive samples, used to evaluate test sensitivity and specificity, consisted of sera with various titres of IgM and IgG. Samples of low volume but high titre were pre-diluted with human plasma known to be negative for the respective virus specific antibodies and viruses (including HIV, hepatitis B virus and hepatitis C virus). Before shipping, the serum panels were tested in duplicate by immunofluorescence assay (IFA), enzyme immunoassay (EIA) and/or immunoblotting (IB). The participating laboratories were advised to dissolve the samples in 100 µl distilled water and to centrifuge for 5 minutes to remove any aggregates before testing. For the EQA of molecular diagnostics each participant received a coded panel of 10 or 33 lyophilised human plasma samples known to be positive or negative for the viral agent in question. The positive samples had been spiked with cell-culture derived and sequence-confirmed strains of virus and contained a range of concentrations between 102 and 107 viral copies per ml. The virus stocks used had been heat inactivated for 1 hour at 56 °C and gamma irradiated with 30 kGy and had been shown to be non-infectious in cell culture. Before shipping, the expected DNA/RNA concentrations in solubilised samples were confirmed by quantitative real-time PCR and, in the case of orthopox viruses (monkeypox strain) and SARS-CoV, virion integrity was assessed by electron microscopy.

The participants in each of the EQA studies were asked to analyze the material provided using the procedures routinely used by them in suspected cases of human infection. They were asked to provide details about the tests, such as the type of the methods used for serological diagnostics (e.g., IFA, EIA, IB or neutralization assay), the protocols and references of the oligonucleotide primers used for PCR, the method used to extract RNA or DNA, and the suppliers and types of commercial kits, if used.

The following two criteria were chosen as the minimum requirements for good overall proficiency: (i) correct identification of the majority of positive samples and (ii) no false positive results for the negative samples. In the case of serological analysis, indeterminate results in positive or negative samples were identified as such and were not used in the evaluation. For molecular detection assays, indeterminate results in positive samples were treated as negative and those in negative samples were treated as positive. This is because tests based on the amplification of nucleic acid do not usually involve indeterminate endpoints and laboratories should be able to resolve unclear results by retesting the sample with a different amplification assay.

Results
The data from the EQA studies conducted through the ENIVD provided a good overview of the diagnostics of those imported, rare and emerging viral infections that have recently become of interest (and a challenge) to expert laboratories involved in public health surveillance both within and outside of Europe. Applying the proficiency criteria, the number of participating laboratories who passed the minimum requirements for successful participation is briefly presented besides other details in table 3. These EQA studies for serological and molecular diagnostics revealed many points that require attention and improvement in the participating laboratories.

Serology
The EQA studies for serological diagnostics revealed that the specificity of the test systems used for the detection of hantavirus- and dengue virus-specific antibodies by the participating laboratories was acceptable (= 97% and = 93% of correctly reported negative results, respectively) [30,31]. However, with only 88% of the negative samples being correctly reported, a lack of specificity for the detection of anti-SARS-CoV antibodies was evident [35]. Generally, for each study, the majority of the participating laboratories achieved good test scores for samples with high antibody concentration but showed poor sensitivity for samples with lower IgM or IgG titres. In particular, the difficulties in diagnosing samples with low IgM titres indicate that there is a considerable risk of overlooking acute infections in patients with low IgM titres. The scores for the correct identification of positive samples by IgM-testing were only 53% for hantavirus, 58% for dengue virus and 64% for SARS-CoV. The in-house and commercial serological tests used in these studies for the detection of antibodies to hantavirus and dengue virus performed with almost equal proficiency and the type of assays used (IFA, EIA or IB) seemed to have little influence on the result. There were, however, clear differences when applied to the serological detection of SARS-CoV, with those laboratories using EIA and/or IB having major problems with regard to sensitivity and specificity. Furthermore, commercial assays performed significantly better than the in-house assays.

Molecular testing
The EQA studies revealed that, with the exception for SARS-CoV, molecular diagnostics showed a poor overall test proficiency than the serological diagnostics. Almost twice as many participating laboratories successfully completed the study for SARS-CoV molecular detection [34] compared to those for dengue virus [32] and viral agents of bioterrorism (filoviruses, Lassa virus and orthopox viruses) [33] (38% for Dengue vs. 45.8% for BT viral agents vs. 87% for SARS-CoV). Although failure was mainly due to a lack of sensitivity, false positive results were also a problem for some laboratories. Such results are particularly troublesome because of the serious public health concerns they can cause in a diagnostic situation. The EQA studies for molecular diagnostics only addressed paramount issues such as sensitivity and the control of contamination while validation of other aspects (e.g. cross-reactivity of primers or control of PCR inhibition) remained the responsibility of each diagnostic laboratory. The use of real-time PCR versus conventional PCR, but not the use of in-house versus commercial PCR, was shown to have a significant impact on a laboratory’s overall sensitivity, especially for detection of dengue virus, filovirus, Lassa virus and orthopox virus. The use of real-time PCR has a positive effect on the diagnostic performance. This may be because real-time PCR is still a relatively new technology normally performed in expert laboratories with a high level of PCR expertise. However, commercial RT-PCR test kits made a significant difference with regard to total sensitivity for SARS-CoV detection. This was clearly the method of choice for good diagnostics, possibly because SARS-CoV is a pathogen with which relatively few participating laboratories have had experience.

Discussion
The results of the EQA studies suggest that there is a need to improve many of the assays in order to improve laboratory diagnostic capabilities. Comparative testing of well-characterised samples provides all participating laboratories with the opportunity to examine their weaknesses and improve methodologies. Several proficiency panels for the EQA of viral diagnostics have been produced for viral pathogens of high prevalence such as HIV, herpes simplex virus, cytomegalovirus and enteroviruses. These panels are offered throughout Europe by commercial organisations such as the UK NEQAS (http://www.ukneqas.org.uk) and INSTAND e.V. (http://www.instand-ev.de), or at the initiative of scientific societies such as the European Union Quality Control Concerted Action formed by the ESCV (http://www.qcmd.org). However, there are still a number of rare (and often not commercially viable) but nevertheless dangerous viral agents that are not addressed by efforts to improve the reliability and quality of the diagnostic output. The ENIVD has begun, for the first time, to generate reference materials for rare viruses that are of public health interest (e.g. for molecular detection of filoviruses, Lassa virus, orthopox viruses and SARS-CoV). Samples are available through the ENIVD for the development and validation of diagnostic tests, and the results generated by the participants in the EQA studies presented here will be a valuable resource for others wishing to establish or improve their own tests. Furthermore, laboratories that require help to improve their diagnostic assays can be supplied with additional diagnostic material or be advised by a competent expert laboratory from the network. It turned out that the diagnostic even of BSL-4 (biosafety level 4) pathogens like filovrius and and Lassa virus or poxviruses can be performed under normal laboratory conditions after an appropriate inactivation is applied. Nevertheless, for a suspected case the ENIVD recommend also to contact one of the BSL-4 expert laboratories for isolation and characterization of the highly infectious pathogen. Given the demand for biological preparedness, regular participation in the EQA programmes will become increasingly important for laboratories worldwide.

A number of EQA programmes in other fields of viral diagnostics have shown that results rapidly improve in subsequent studies [36]. Presently, the ENIVD is conducting a second EQA run for the molecular diagnostics of orthopox viruses, this time taking into consideration how laboratories prevent PCR inhibition by various inhibitory factors that might cause quality deterioration in serum or tissue samples from clinical cases or in samples from environmental sources [37]. This second run shows preliminary a significant improvement of the diagnostic performance for the participating laboratories as compared to the first EQA run (unpublished data).

In May 2005 the European Centre for Disease Prevention and Control (ECDC) became fully operational, and this represents a new milestone in measures to defend against and prevent threats to public health – both natural or deliberate – in the European Community [38]. The scientific expertise and information made available through various disease specific EU networks like the ENIVD can be brought together by the ECDC to fulfil its important tasks. Furthermore, the existing network structures can be used to expand the international role played by the EU in tackling diseases, particularly in neighbouring countries, and its role in the global action to control and respond to serious outbreaks or threats. The benefits of such close cooperations with existing networks could be shown for the immediate development and provision of reliable diagnostic tools in response to SARS, where the Global Outbreak and Response Network of the WHO (GOARN) and the ENIVD worked together to enable laboratories to perform the appropriate diagnostics. The ENIVD is planning to expand its network structure by inviting additional laboratories from EU-candidate countries and neighbouring Eastern countries to participate as permanent members.

Acknowledgements
The ENIVD was funded by the EC DG SANCO under the programme AIDS and other communicable diseases grant No. SI12.299717 (2000CVG4-26). The EQA studies received excellent assistance from A. Teichmann. We thank Antonio Tenorio and William Hall for personal communications as well as Marcel Müller and Stephen Norley for critical reading of the manuscript.