In 2024, Israel experienced its largest West Nile Virus (WNV) outbreak, reporting 934 cases. Diagnosis primarily relies on serological testing for IgM antibodies; however, cross-reactivity with other flaviviruses and prolonged IgM persistence complicate interpretation. Molecular testing is less utilized due to concerns about the short duration of viremia and potential false negatives.

To evaluate the diagnostic reliability and persistence of WNV molecular testing across different sample types compared to serological testing, leveraging the extensive sample collection during the 2024 outbreak in Israel.

Samples from 919 WNV cases, including whole blood (WB), serum, urine, and cerebrospinal fluid (CSF) were evaluated. WNV RNA, IgM antibodies and IgG avidity were tested on all sample types, serum and CSF and serum, respectively.

WNV RNA was detected in 91 % of WB samples, and observed up to 52 days post-symptom onset. Detection rates were lower in serum (82 %), urine (71 %), and CSF (53 %), with shorter detection windows. Viral concentrations were highest in urine, followed by WB with serum, and CSF, the lowest. IgM antibodies were present in 83 % of serum and 86 % of CSF samples. RNA detection rates were comparable between hospitals (91 %) and health maintenance organizations (HMOs) (92 %), but IgM positivity was significantly higher in hospitals (91 %) than in HMOs (69 %).

Molecular testing on WB offers the highest sensitivity and longest detection window for WNV RNA. Molecular diagnosis enhances accuracy, facilitates earlier detection, and improves clinical and public health response strategies compared to serological diagnosis.