Evaluation of the diagnostic efficacy of a reagent kit for <i>in vitro</i> diagnosis of West Nile fever using reverse transcription polymerase chain reaction with fluorescent probe-based detection

West Nile fever is a vector-borne zoonotic arbovirus infection with natural foci. Its clinical course is similar to that of acute febrile syndrome, and severe cases may result in neuroinvasive disease. Several genetic lineages (1, 2, and 4) of the West Nile virus (WNV) with different pathogenicity for humans are circulating in the Russian Federation. Therefore, it is an urgent task to develop a diagnostic reagent kit for differentiating between WNV genetic lineages and to implement the kit in clinical laboratory practice.The aim of the study was to conduct technical and clinical tests and evaluate the quality, efficacy, and safety of the Ampligen-WNV-genotype-1/2/4 diagnostic reagent kit for detecting WNV RNA and differentiating between WNV genetic lineages 1, 2, and 4 by reverse transcription polymerase chain reaction (RT-PCR) with fluorescent probe-based detection.Materials and methods. The authors determined the diagnostic sensitivity and specificity of the Ampligen-WNV-genotype-1/2/4 reagent kit (Volgograd Research Institute for Plague Control, Russia) by real-time RT-PCR with 216 clinical samples and 204 biological samples. Sanger sequencing was used as a reference method. Statistical analysis of clinical test results was carried out in accordance with the Russian national standard for clinical laboratory tests (GOST R 53022.3-2008).Results. When tested with the Ampligen-WNV-genotype-1/2/4 reagent kit, real-time RT-PCR demonstrated the analytical sensitivity of 1×104  GEq/mL for the detection of WNV cDNA of genetic lineages 1, 2, and 4. The assessment of its analytical specificity showed no positive results for cDNA samples of heterologous viruses at a concentration of 1×106  GEq/mL. The diagnostic sensitivity with the reagent kit was at least 98.5%, and the diagnostic specificity was at least 99%, with 90% confidence levels for both parameters.Conclusions. The Ampligen-WNV-genotype-1/2/4 reagent kit can be recommended for use in clinical laboratory diagnostics to detect WNV RNA and differentiate between WNV genetic lineages 1, 2, and 4.