# One-Year Monitoring of the Evolution of SARS-CoV-2 Omicron Subvariants Through Wastewater Analysis (Central Italy, August 2023–July 2024)

## TL;DR

This study used wastewater analysis in Italy to track the evolution of SARS-CoV-2 Omicron subvariants over a year, showing wastewater surveillance can detect viral changes earlier than clinical data.

## Contribution

The study demonstrates wastewater surveillance as an effective early warning system for SARS-CoV-2 subvariant evolution during low infection periods.

## Key findings

- Wastewater surveillance detected three distinct Omicron subvariant phases (XBB.*, BA.2.86/JN, KP.*) in Central Italy from August 2023 to July 2024.
- Wastewater data revealed higher viral lineage diversity and detected subvariants like BA.2.86* weeks to months before clinical identification.
- The KP.* subvariant, particularly KP.3, became dominant in wastewater by July 2024, highlighting wastewater's role in tracking viral dynamics.

## Abstract

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.