BACKGROUND: In recent decades, sporadic cases and outbreaks in humans of West 
Nile virus (WNV) infection have increased. Serological diagnosis of WNV 
infection can be performed by enzyme-linked immunosorbent assay (ELISA), 
immunofluorescence assay (IFA) neutralization test (NT) and by 
hemagglutination-inhibition assay. The aim of this study is to collect updated 
information regarding the performance accuracy of WNV serological diagnostics.
METHODOLOGY/PRINCIPAL FINDINGS: In 2011, the European Network for the 
Diagnostics of Imported Viral Diseases-Collaborative Laboratory Response Network 
(ENIVD-CLRN) organized the second external quality assurance (EQA) study for the 
serological diagnosis of WNV infection. A serum panel of 13 samples (included 
sera reactive against WNV, plus specificity and negative controls) was sent to 
48 laboratories involved in WNV diagnostics. Forty-seven of 48 laboratories from 
30 countries participated in the study. Eight laboratories achieved 100% of 
concurrent and correct results. The main obstacle in other laboratories to 
achieving similar performances was the cross-reactivity of antibodies amongst 
heterologous flaviviruses. No differences were observed in performances of 
in-house and commercial test used by the laboratories. IFA was significantly 
more specific compared to ELISA in detecting IgG antibodies. The overall 
analytical sensitivity and specificity of diagnostic tests for IgM detection 
were 50% and 95%, respectively. In comparison, the overall sensitivity and 
specificity of diagnostic tests for IgG detection were 86% and 69%, 
respectively.
CONCLUSIONS/SIGNIFICANCE: This EQA study demonstrates that there is still need 
to improve serological tests for WNV diagnosis. The low sensitivity of IgM 
detection suggests that there is a risk of overlooking WNV acute infections, 
whereas the low specificity for IgG detection demonstrates a high level of 
cross-reactivity with heterologous flaviviruses.