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Background West Nile virus (WNV) is increasingly reported in Italy, but its true prevalence remains uncertain. People living with HIV (PLWH) may be especially vulnerable given their potential risk of developing neuroinvasive disease. This study aimed to assess the seroprevalence and risk factors of WNV and Usutu virus (USUV) among PLWH in our cohort.Methods This retrospective single-center study included all PLWH routinely followed at the Brescia HIV Outpatient Clinic with at least one serum sample collected in 2022 and stored in the Brescia sero-repository. Serum samples were screened for WNV antibodies using first an ELISA test, all positive or borderline samples for IgG and IgM were then confirmed by immunofluorescence assay. In a second time, all positive samples were further characterized by microneutralization test against both WNV and USUV for differential diagnosis. Demographic, clinical, and HIV-related variables were extracted from medical records and systematically recorded. Associations between variables and seropositivity were evaluated by univariate logistic regression.Results Among 2,843 PLWH, the median age was 55 years; 76.2% were male and 87.2% were of Italian origin. Most participants had undetectable HIV-RNA and no history of ART interruption (94.2%). The mean CD4/CD8 ratio was 1.01 (SD±0.59). WNV infection was identified in 3.0% of participants, USUV infection in 0.6%, and co-infection in 0.2%. Fever was reported in 3.1% of confirmed cases, and no neuroinvasive disease was observed. Compared with WNV-seronegative PLWH ( table 1), individuals with positive neutralization assays for WNV or USUV were less frequently male (p=0.001) and differed significantly by geographic origin (p<0.001). WNV-Seropositivity was more common among participants born out of Europe, whereas Italian origin predominated among seronegative individuals. In logistic regression analyses, geographic origin was strongly associated with seropositivity. Compared with Italian-origin participants, those from Africa (OR 18.94, 95% CI 11.53–31.09), Asia (OR 20.41, 95% CI 9.18–45.36), and the Americas (OR 16.90, 95% CI 8.60–33.21) had significantly higher odds of WNV or USUV seropositivity (all p<0.001). Male sex was associated with lower odds of seropositivity (OR 0.51, 95% CI 0.34–0.77; p=0.001).Conclusions To the best of our knowledge, this is first study assessing WNV and USUV seroprevalence in PLWH. Our findings indicate a comparatively higher seroprevalence in this population than that reported in analogous Western blood donor cohorts. Most confirmed cases were asymptomatic and occurred predominantly in individuals born in endemic regions. These data suggest that migration from endemic areas represents the principal determinant of WNV exposure among PLWH in our cohort. Furthermore, HIV infection does not appear to significantly influence the clinical course of West Nile virus infection.Abstract SC7 Table 1Comparison of neutralization assay results between WNV or USUV cases and seronegative PLWH (Acronym: WNV, West Nile virus; USUV, Usutu virus; *On stable ART, defined as no treatment discontinuation exceeding one year since ART initiation)