Urine: A Pitfall for Molecular Detection of Toscana Virus? An Analytical Proof-of-Concept Study

Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical features with other neuro-invasive viral infections. Nowadays, the reference test for direct diagnosis in the acute phase of TOSV infection is the PCR based method for detecting TOSV in cerebrospinal fluid and/or plasma, serum, or blood. Although poorly employed, urine is another helpful biological matrix for TOSV detection. Urine is a matrix rich in PCR inhibitors that affect PCR efficiency; consequently, false negatives could be generated. To investigate the potential effect of urine PCR inhibitors on TOSV detection, we compared undiluted and diluted urine using 10-fold series of spiked TOSV. The results showed a significant improvement in TOSV detection performance in diluted urine (1 TCID₅₀ vs. 1 × 10⁴ TCID₅₀ limit of detection and 101.35% vs. 129.62% efficiency, respectively, in diluted and undiluted urine). In conclusion, our data provide preliminary important insights into the use of diluted urine to limit the impact of the inhibitory effects of urine on the detection of TOSV in RT-PCR-based approaches.