ENHANCING UNDERSTANDING OF SARS-CoV-2 VARIANTS USING WASTEWATER ANALYSIS: AN In-DEPTH 18-MONTH INVESTIGATION

Aim of the Study: Since its onset in 2020, the COVID-19 pandemic has rapidly spread across the world, resulting in a significant increase in morbidity and mortality rates. The emergence of several SARS-CoV-2 variants has added to the concern of global health authorities. These variants contain unique sets of mutations that provide higher transmissibility or, in some cases, more severe symptoms in infected individuals. Wastewater-Based Epidemiology (WBE) has proved to be an effective tool for understanding and monitoring the COVID-19 pandemic. The analysis of waste water provides valuable insights into the extent and progression of a disease outbreak within a specific area. Additionally, this method enables tracking the emergence and spread of new viral variants. Recognizing the potential of WBE technology, the European Commission recommended establishing systematic surveillance of SARS-CoV-2 and its variants in wastewater across all EU regions. Since October 2021, Italy has been conducting monthly “flash surveys” in all regions to monitor the SARS-CoV-2 variant’s spread in the community. In this study, we present the results of 18 months of wastewater monitoring in Italy. Methods: During the period spanning October 2021 to March 2023, a total of 2731 samples of wastewater were collected from 167 water treatment plants located in 18 regions and 2 autonomous provinces across Italy. These samples underwent analysis to detect the presence of SARS-CoV-2 variants. Specifically, the samples underwent amplification using previously de scribed nested RT-PCR (1), able to amplify a long fragment (1592 bps) of the spike region to detect distinctive mutations of SARS-CoV-2 variants. Positive PCR amplicons were sequenced by both Sanger and Next-Generation (NGS) techniques. Results and Conclusions: Overall, 654 samples were successfully sequenced. The study found shifts in the predominant viral variants in wastewater over the 18-month monitoring period, in line with the epidemiological conditions. In fact, from October 2021 to December 2021, the only variant identified was the Delta. Starting January 2022, the Omicron variant was dominant, with several sublineages emerging periodically: 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, and CM*. The NGS ap proach enabled the identification of less prevalent viral variants that might otherwise be missed by Sanger sequencing. These findings demonstrate that WBE can be used to monitor the evolution and spread of SARS-CoV-2 at the population level. The results of this study emphasize the importance of wastewater surveillance for SARS-CoV-2 control strategies and the need for continued monitoring of viral strains in both clinical and environmental settings. Acknowledgements This study is supported by the EU Commission (Grant Agreement 060701/2021/864481/ SUB/ENV.C2) and partially by the European Union – NextGenerationEU within the National Recovery and Resilience Plan (NRRP) PE13 INF-ACT. Reference 1. La Rosa G, Mancini P, Bonanno Ferraro G, Veneri C, Iaconelli M, Lucentini L, Bonadonna L, Brusaferro S, Brandtner D, Fasanella A, Pace L, Parisi A, Galante D, Suffredini E. 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 Res. 2021 Jun 1; 197: 117104. doi: 10.1016/j.watres.2021.117104.