SARS-CoV-2 VARIANTS CIRCULATION IN ITALY: A 30-MONTH WASTEWATER-BASED STUDY

Background: Wastewater-based epidemiology (WBE) is emerging as an important non-invasive method for tracking viral infections such as SARS-CoV-2. Analysis of wastewater provides insight into viral prevalence and spread, especially when clinical testing is scarce. The European Commission’s recommendation for systematic surveillance of SARS-CoV-2 in wastewater underscores its importance. Since October 2021, Italy’s implementation of monthly “flash surveys” illustrates the value of WBE in monitoring virus spread and circulating variants. This study presents the results of 30 months of wastewater monitoring of SARS-CoV-2 variants in Italy. Methods: Between October 2021 and February 2024, 3906 wastewater samples were collected throughout Italy. The samples were collected from 167 wastewater treatment plants located in 18 regions and 2 autonomous provinces. These samples were analysed to detect the presence of SARS-CoV-2 variants. Specifically, the samples were subjected to amplification using a previously described nested RT-PCR method (1), which enabled the amplification of a long fragment (~1600 bps) of the spike region, thus allowing the identification of different mutations of SARS-CoV-2 variants. The positive PCR amplicons were sequenced using both Sanger (single sample) and Next-Generation Sequencing (pooled samples by Regions/A.P). Since April 2023, following the cessation of funding, only Sanger sequencing has been carried out. Results and Conclusions: In total, 979 samples were successfully sequenced. The study observed shifts in the dominant viral variants in wastewater over the 30-month monitoring period, reflecting the epidemiological conditions. In particular, between October 2021 and December 2021, the Delta variant was the only variant identified. In January 2022, Omicron became the dominant variant, associated with the emergence of several sublineages. The variants identified by then were: BA.1, BA.1.1, BA.2, BA.4, BA.5, BQ1, BQ.1.1, BA.2.75 (including BN.1* and CH.1.1), XBB.1, XBB.1.6, XBB.1.5/XBB.1.9, CM.7, XBB.2.3, GL.1, JN.1, and EG.5. Since December 2023 the JN.1 variant became prevalent in our country. The NGS approach enabled the identification of less prevalent viral variants otherwise missed by Sanger sequencing. These findings underscore the pivotal role of wastewater surveillance in enhancing our understanding of circulating viral strains, serving as a complementary tool to clinical approaches. Acknowledgements: This study was partially supported by the EU Commission (Grant Agreement 060701/2021/864481/SUB/ENV.C2) and partially by EU funding within the NextGeneration EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, PE13 INF-ACT). Reference La Rosa et al., 2021. Rapid screening for SARS-CoV-2 variants of concern in clinical and environmental samples using nested RT-PCR assays targeting key mutations of the spike protein. Water research, 197, 117104. https://doi.org/10.1016/j.watres.2021.117104.