Chikungunya fever is an arboviral disease transmitted by Aedes mosquitoes. The disease was first described in 1953 in Tanzania and has since become endemic in parts of Africa, Southeast-Asia and on the Indian sub-continent [1-5]. Chikungunya virus (CHIKV) is an alphavirus belonging to the family Togaviridae which has a single-stranded RNA genome, a 60-70 nm diameter capsid and a phospholipid envelope. The Makonde name 'chikungunya' means 'that which bends up' and refers to the stooped posture many patients develop as a result of painful and invalidating polyarthralgia commonly associated with the disease [1]. While in the past chikungunya was not generally considered to be a life-threatening disease, recently a substantial number of deaths (254) have been attributed, directly or indirectly, to this virus [2]. Some patients with a confirmed CHIKV infection developed severe clinical signs (i.e. neurological signs or fulminant hepatitis) that justified hospitalisation in an intensive care unit. Cases of neonatal encephalopathy and major algic syndrome associated with vertical transmission of the virus were also reported [3,4].

CHIKV has been imported to Europe (France, Germany, Switzerland, Italy and Norway) by infected travellers returning from tropical areas with high incidence rates, and Aedes albopictus has been introduced into several European countries (Albania, Belgium, Bosnia, Croatia, France, Greece, Italy, Montenegro, The Netherlands, Serbia, Slovenia, Spain and Switzerland)* [6-9]. So far, none of the imported cases has resulted in subsequent transmission of the virus. But, it has been suggested that if viraemic patients arrive in Southern Europe during the summer they could cause a European outbreak [8,10]. Currently, an outbreak in Northern Italy has attracted much attention as the first autochthonous CHIKV outbreak in Europe [11].

For disease control in the borderless European Union early recognition and efficient diagnostic methods are indispensable. Therefore, external quality assurance (EQA) studies of diagnostic methods are important to increase the awareness of emerging diseases and to establish and evaluate appropriate detection assays. In June 2007, the European Network for Diagnostics of Imported Viral Diseases (ENIVD) started an EQA analysis of both serological and molecular methods used for CHIKV detection. Here we give preliminary results regarding the laboratory capacity and diagnostic quality for detection of CHIKV infections in Europe.

Methods

Since sensitivity and specificity of the often used in-house serological tests (immunfluorescence assay, plaque reduction neutralisation assay, hamaglutination inhibition assay, ELISA) and PCR (real-time PCR, nested-PCR) [1] are poorly evaluated, two proficiency panels with test samples were generated to assess the quality of CHIKV diagnostics. For the EQA of serological diagnostics, each participating laboratory received a panel of 12 coded lyophilised samples comprising samples positive for antibodies against CHIKV and negative controls. All used human sera were diluted before being freeze dried in aliquots of 100 µl as described previously [12]. For the EQA of molecular diagnostics, panels were generated by diluting inactivated virus stock solutions. The panels comprise samples with different CHIKV strains (from Réunion, India, Seychelles, Mauritius and East-Africa) including a dilution series for evaluation of the assay sensitivity, and negative control samples. Supernatants from chikungunya infected cell cultures were inactivated by heat and gamma irradiation before being aliquoted and freeze dried as described previously [13]. The participants in each of the EQA parts were asked to analyse the material provided using the procedures routinely used by them in suspected human cases. Assay details, such as the type of methods used, protocols, references and suppliers of commercial kits (if any), were requested. Rates of <70% correct classified results were considered as a bad performance.

This EQA analysis was advertised as a study on diagnostic proficiency run by the ENIVD, including publication of the results in a comparative and anonymous manner. Participation was open and free of charge to all laboratories performing CHIKV diagnostics. Selection of invitees was based on the register of ENIVD members, on a further announcement done by the World Health Organization (WHO) as well as on the contributions of the participants to the literature relevant to this topic.

Preliminary results

A total of 24 laboratories from 15 European countries – Austria (1), Belgium (1), Denmark (1), France (3), Germany (5), Greece (1), Ireland (1), Italy (2), Norway (1), Portugal (1), Slovenia (1), Spain (1), Sweden (1), Switzerland (2) and The Netherlands (2) – participated in at least one part of EQA (20 expert laboratories for PCR and 18 expert laboratories for serology) by sending back their results within 69 days after receiving the samples.
PCR
The analysis of the results revealed considerable variations in sensitivity and specificity for the PCR. While most laboratories showed sufficient sensitivity and specificity (16/20) some (4) had general problems with low sensitivity and specificity showing failures in detection of viral loads well above any known detection limit (>29,000 RNA copies per millilitre) and/or detection of different CHIKV strains. However, only one laboratory showed a false positive result in a negative sample indicating contamination during the PCR processing.
Serology
For serology we found even greater differences between the results of the various laboratories, probably partly due to the different assays used. Of 14 laboratories that performed analysis for IgM/IgG, eight showed a good result for both IgM and IgG while others detected only sera with a high IgM titre. Of the four laboratories that did only serology for IgG three showed a rather good performance whereas one laboratory had a low sensitivity and specificity and showed <70% of correct results. Fortunately, none of the participating laboratories showed false-positive reactions from cross-reactivity with antibodies against viruses other than CHIKV.

Conclusion

Preliminary results of this first EQA analysis revealed considerable variations in both availability and performance of CHIKV diagnostics across Europe. Capacity for detection of CHIKV seems to be much lower in Eastern Europe compared to the West. Of the 10 Member States that joined the European Union in 2004, only one laboratory in Slovenia took part in the evaluation. In the rest of Europe, however, we found a wide distribution of laboratories performing CHIKV diagnostic, even though some did less well than others.

The great variation in the performance of the different laboratories presents a similar picture as found in EQA studies performed for other rare and/or exotic viruses like Hanta or Dengue [14-16]. This was the first EQA study for CHIKV. Several laboratories started to diagnose it with lack of experience and missing assay evaluation, which may explain the great diversity of the results. The final evaluation will show which of the PCR and serology assays used perform best with highest sensitivity and specificity. All laboratories with a bad performance were immediately informed and improvements suggested.

A possible solution to overcome the mentioned gaps is to share diagnostic capacity within Europe (i.e. send specimens to expert laboratories abroad), as it is done in the case of other rare diseases. The task of coordinating this belongs to ENIVD. Whether diagnostic capacity regarding CHIKV needs to be enlarged in the future will very much depend on the development of the epidemic situation. Growing risk of infection for people travelling to new and/or enlarged CHIKV endemic areas as well as the spread and establishment of Aedes albopictus in Europe may considerably increase the demand for efficient diagnostic of chikungunya fever in the coming years.

Information on ENIVD and CHIKV is available on the public website at: http://www.enivd.org
Users can also find there an overview on all diagnostic tests performed by the network, recommendations for laboratory procedures and fact sheets of important emerging and re-emerging viral diseases.

Acknowledgements:
The EQA analysis was performed by the European Network for Diagnostics of Imported Viral Diseases (ENIVD) presently funded by the European Commission’s Directorate-General for Health and Consumer Protection (DG SANCO) under the programme AIDS and other communicable diseases Grant No. 2004206. We thank Christian Drosten, Hervé Zeller, Isabelle Schuffenecker and Remi Charrel for providing material to perform this EQA study.

* Authors correction
In the second paragraph, Portugal was erroneously listed among European countries where Aedes albopictus has been introduced. This was corrected on 27 September 2007.