Long-Term Wastewater Genomic Surveillance Reveals SARS-CoV-2 Variant Dynamics in Italy from 2021 to 2025

Abstract

Long-term genomic surveillance of SARS-CoV-2 remains important as routine clinical testing and sequencing decline over time after the pandemic. We describe the temporal dynamics, methodological adaptation, and operational continuity of Italy’s national wastewater variant surveillance from October 2021 to December 2025. Across 51 monthly surveys, 6,278 wastewater samples collected from 167 wastewater treatment plants in 18 Regions and 2 Autonomous Provinces were analysed by RT-qPCR and targeted spike sequencing using short- and long-amplicon Sanger and Oxford Nanopore Technology approaches. Overall, 1,186 samples yielded a successful long amplicon and an interpretable Sanger sequence, while 248 regional pools were characterized by ONT during the funded surveillance phase from October 2021 to March 2023. The data documented the replacement of Delta by Omicron, followed by the circulation of BA.1*, BA.2* and BA.4/BA.5* and the subsequent emergence of BQ*, XBB*, EG.5*, and JN.1* lineages, with periods of persistence and co-circulation. The analytical workflow was adapted as lineage-defining mutations changed. Since March 2023, surveillance continued voluntarily through December 2025 using only long-amplicon Sanger sequencing. A qualitative comparison with national periodic flash surveys to estimate the prevalence of SARS-CoV-2 variants on biological samples showed that the two systems broadly captured the same succession of major lineage transitions, although the variant prevalence estimates and wastewater lineage proportions were not directly comparable. These findings support the feasibility of a targeted and adaptable strategy for sustaining nationwide wastewater genomic surveillance across emergency and post-emergency phases and for complementing clinical surveillance when routine testing and sequencing are reduced.