Wastewater surveillance has proven to be a cost-effective, non-invasive method for monitoring the spread and evolution of SARS-CoV-2, yet its value during today’s low-incidence phase is still being defined. Between August 2023 and July 2024, 42 composite wastewater samples were collected in Perugia, Italy and analyzed using RT-qPCR and whole-genome sequencing to identify circulating SARS-CoV-2 lineages. In parallel, clinical samples (respiratory tract samples) were collected and analyzed, allowing for direct comparisons to confirm the robustness of the wastewater findings. The sewage viral loads ranged from 8.9 × 10⁵ to 4.9 × 10⁷ genome copies inhabitant⁻¹ day⁻¹, outlining two modest community waves (September–December 2023 and May–July 2024). Sequencing resolved 403 Omicron lineages and revealed three successive subvariant phases: (i) XBB.* dominance (August–October 2023), when late-Omicron XBB subvariants (mainly EG.5.* and XBB.1.5) accounted for almost all genomes; (ii) a BA.2.86/JN surge (November 2023–March 2024), during which the BA.2.86 subvariant, driven mainly by its JN descendants (especially JN.1), rapidly displaced XBB.* and peaked at 89% in February 2024; and (iii) KP.* takeover (April–July 2024), with JN.1-derived KP subvariants rising steadily and KP.3 reaching 81% by July 2024, thereby becoming the dominant lineage. Comparisons of data from wastewater and clinical surveillance demonstrated how the former presented a much higher diversity of circulating viral lineages. Importantly, some subvariants (including BA.2.86*) were detected in wastewater weeks to months prior to clinical identification, and for longer periods. Taken together, the obtained data validated wastewater surveillance as an effective early warning system, especially during periods of low infection prevalence and/or limited molecular testing efforts. This methodology can thus complement clinical surveillance by offering valuable insights into viral dynamics at the community level and enhancing pandemic preparedness.

Background/Objectives: The prevalence of Hepatitis C Virus (HCV) among men who have sex with men (MSM) is not negligible; however, data from Italy—especially regarding MSM who are not infected with Human Immunodeficiency Virus (HIV)—are limited. We report data from an HCV screening programme targeted at MSM in Rome, starting in 2019 in two hospital settings and in four urban community-based (CB) settings run by non-governmental organizations (NGOs). Methods: Adult MSM (>18 years old) who presented for HIV or sexually transmitted infection testing, or who attended CB activities, were invited to undergo a free-of-charge rapid HCV antibody test (OraQuick HCV^®), after providing informed consent. For all participants, demographic, clinical and behavioural data were collected using an anonymous questionnaire for all participants. Free confirmatory standard serology tests were offered for those found reactive at a rapid HCV test. Individuals with confirmed chronic HCV infection were referred through a dedicated “fast track” pathway for further clinical and laboratory assessment and direct-acting antiviral agents (DAAs) treatment according to the national treatment guidelines. Results: Between July 2019 and July 2023, 2714 MSM agreed to be screened for HCV infection. The median age was 36 years (interquartile range, IQR = 29–46), 91.0% were Italians, 58.0% enrolled in the two clinical centres and 10.7% reported living with HIV (people living with HIV, PLWH). Overall, 9 (0.33%) MSM tested reactive for HCV-specific antibodies using a rapid test. Eight MSM were retested and seven were confirmed to have chronic HCV infection (HCV viremia range: 8 × 10³–23 × 10⁶ IU/mL). The prevalence of confirmed cases was 0.26% (7/2714; 95%CI: 0.10–0.53) and was higher in PLWH compared to those not reporting HIV infection (1.04% vs. 0.17, p = 0.03). Four of seven confirmed HCV cases attended the STI clinic. All confirmed HCV cases reported high-risk behaviours for HCV infection and/or history of sexual transmitted infection (STI). Bening a PLWH (OR = 6.30) and current/former IDU (O = 17.02) resulted in being significantly associated with HCV infection. Other risk factors such as fisting, groupsex, chemsex and condomless anal intercourse were more common in the HCV case (OR > 2), but lacked statistical significance, likely due to small sample size. All seven individuals were linked to care, clinically assessed and started on DAAs treatment, achieving sustained viral response (SVR) in all cases. Conclusions: These data suggest the feasibility and potential effectiveness of a preventive programme targeting MSM living in Rome, combining HCV screening, case finding and prompt linkage to care. HCV prevalence in the screened population was lower than anticipated, although it is significantly higher in PLWH and in those with high-risk behaviours. Considering this condition of low prevalence of HCV infection among MSM in Italy, a targeted screening in PLWH and in individuals with high-risk behaviours may be more effective to achieve HCV eradication than universal screening in MSM.

West Nile virus (WNV) is a flavivirus whose cycle involves mosquitoes and avian hosts that occasionally may infect horses and humans as “dead‐end hosts.” Human infection is mostly asymptomatic, sometimes leading to febrile or neuroinvasive disease. In 2025, Italy experienced its largest epidemic with an unprecedented geographic spread (79 provinces; 52% of cases in central Italy). Here we report whole‑genome sequencing and phylogeographic analysis of 39 WNV lineage 2 samples collected in Lazio and Campania regions between July and September 2025 from humans and animal samples. All 2025 strains belonged to a Southern Italian clade that diversified around 2020, with evidence of earlier circulation in 2024. All the 2025 genomes shared a substitution that lies in envelope protein domain III (K399R), potentially influencing interactions with the viral membrane. The same mutation was previously detected in Eastern European strains, leading to the hypothesis of introductions in Italy via migratory birds from these regions. Phylogeography identified strong viral dispersal routes within Central Italy supporting a pattern of short‑range spread and local overwintering. Overall, results highlight the roles of ecological conditions and cross‑border transmission dynamics in shaping the 2025 Italian epidemic, underscoring the need for integrated genomic/ecological surveillance.

Wastewater-based surveillance has emerged as a powerful approach for population-level monitoring of pathogen circulation in a timely and non-invasive manner. In this study, shotgun metagenomic sequencing was applied to wastewater samples collected from a hospital (HP), a university campus (UN), and a wastewater treatment plant (WTP). Viral sequences were taxonomically classified using Kraken2. Specifically, HP samples showed the highest viral richness, followed by WTP and UN samples (HP vs. UN, p = 0.0003; WTP vs. UN, p = 0.0018). Using Jaccard distance, significant differences were observed between WTP and UN (R² = 0.181, p < 0.001), WTP and HP (R² = 0.159, p < 0.001), and UN and HP (R² = 0.223, p < 0.001), and similarly, for Sørensen–Dice dissimilarity: WTP vs. UN (R² = 0.238, p < 0.001), WTP vs. HP (R² = 0.212, p < 0.001), and UN vs. HP (R² = 0.307, p < 0.001). Human-associated viral families were detected across all sources, predominantly Poxviridae, Orthoherpesviridae, Polyomaviridae and Circoviridae. Furthermore, the taxonomic composition of indirectly associated viruses, mainly Anelloviridae and Crassvirales, was examined. Overall, these findings support the potential of wastewater metagenomics as a reliable tool for monitoring viral diversity within environmental and public health contexts, although further research is needed to establish its operational utility for routine surveillance applications within a One Health framework.

Injection drug use represents an important contributor to hepatitis C virus (HCV) transmission, hence therapeutic communities (TCs) are promising points of care for the identification and treatment of HCV-infected persons who inject drugs (PWIDs). We evaluated the effectiveness and efficacy of an HCV micro-elimination program targeting PWIDs in the context of a drug-free TC; we applied the cascade of care (CoC) evaluation by calculating frequencies of infection diagnosis, confirmation, treatment and achievement of a sustained virological response (SVR). We also evaluated the risk of reinfection of PWIDs achieving HCV eradication by collecting follow-up virologic information of previously recovered individuals and eventual relapse in drug use, assuming the latter as a potential source of reinfection. We considered 811 PWIDs (aged 18+ years) residing in San Patrignano TC at the beginning of the observation period (January 2018–March 2022) or admitted thereafter, assessing for HCV and HIV serology and viral load by standard laboratory procedures. Ongoing infections were treated with direct-acting antivirals (DAA), according to the current national guidelines. Out of the 792 individuals tested on admission, 503 (63.5%) were found to be seropositive for antibodies against HCV. A total of 481 of these 503 individuals (95.6%) underwent HCV RNA testing. Out of the 331 participants positive for HCV RNA, 225 were ultimately prescribed a DAA treatment with a sustained viral response (SVR), which was achieved by 222 PWIDs (98.7%). Of the 222 PWIDs, 186 (83.8%) with SVR remained HCV-free on follow-up (with a median follow-up of 2.73 years after SVR ascertainment). The CoC model in our TC proved efficient in implementing HCV micro-elimination, as well as in preventing reinfection and promoting retention in the care of individuals, which aligns with the therapeutic goals of addiction treatment.

Background/Objectives: HCV prevalence among men who have sex with men (MSM) is not negligible, but information in Italy is scarce, particularly for HIV-uninfected MSM. We report data from an HCV screening program targeted to MSM in Rome started in 2019 in two hospital settings and in four urban community-based (CB) settings run by NGOs. Methods: Adult MSM (>18 years old) presenting for HIV or Sexually transmitted infection testing or attending CB activities, have been invited to undergo, after providing informed consent, a free-of-charge rapid HCV Antibody test (OraQuick HCV®). For all participants, demographic, clinical and behavioural data using an anonymous questionnaire were collected. Free confirmatory standard serology tests were offered for those found reactive at rapid HCV test and then individuals with confirmed chronic HCV-infection, were referred though a dedicated “fast track” pathway for further clinical and laboratory assessment and DAA-treatment according to the national treatment guidelines. Results: From July 2019 and July 2023, 2.714 MSM agreed to be screened for HCV infection. Median age was 36 years (IRQ=29-46), 91.0% were Italians, 58,0% enrolled in the two clinical centers and 10,7% reported to be person living with HIV (PLWH). Overall, 9 (0,33%) MSM were found to be reactive for HCV-Ab at rapid test. Eight MSM were retested and 7 were confirmed as having chronic HCV infection (HCV viremia range: 8x103-23x106 IU/mL). The prevalence of confirmed cases was 0.26% (7/2,714; 95%CI: 0.10-0.53), higher in PLWH compared to those not reporting HIV infection (1.04% vs. 0.17, p=0.03). High-risk behaviours for HCV infection and/or history of Sexual Transmitted infection (STI) were recorded in all HCV confirmed cases. All seven persons were linked to care, clinically assessed and started DAA treatment, with sustained viral response (SVR) in all cases. Conclusions: These data suggest feasibility and potential effectiveness of a preventive program targeting MSM living in Rome, which combines HCV screening, case finding and linkage to prompt care. HCV-prevalence in the screened population, however, was quite low, although it is significantly higher in PLWH and in those with high-risk behaviours, suggesting that targeted screening of PLWH and individuals with high-risk behaviours may be more effective to achieve HCV eradication than universal screening.