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

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.

Keywords: RT-qPCR; SARS-CoV-2; next-generation sequencing; viral variants; wastewater surveillance.

Figure 1

Quantitative trend analysis of SARS-CoV-2 viral load as determined by wastewater analysis: Temporal trends of SARS-CoV-2 viral load (expressed as g.c. inh⁻¹ d⁻¹) were measured by RT-qPCR in RNA samples extracted from urban wastewater collected between 9 August 2023 and 1 July 2024 from the wastewater treatment plant of Pian della Genna (Perugia, Italy).

Figure 2

Relative abundances of the main SARS-CoV-2 lineages circulating in the studied area, as assessed by wastewater analysis: SARS-CoV-2 lineages in wastewater samples collected between 9 August 2023 and 1 July 2024 from the wastewater treatment plant of Pian della Genna (Perugia, Italy) were determined using amplicon next-generation sequencing. Lineage identification and calculation of relative abundances at each timepoint were carried out using Freyja’s tool. For clarity, the main lineages found are shown in different panels, with the first and second depicting a detailed division of Omicron XBB lineages, the third grouping all Omicron BA.2.86 lineages together (to highlight their appearance and the gradual replacement of XBB* lineages), and the fourth reporting all other lineages that were not classified in the previous two groups.

Figure 3

Relative abundances of the main circulating BA.2.86 lineages, as assessed by wastewater analysis: Wastewater samples collected between 18 October 2023 (date of first appearance of Omicron BA.2.86) and 1 July 2024 from the wastewater treatment plant of Pian della Genna (Perugia, Italy) were analyzed by amplicon next-generation sequencing, and lineages were identified with Freyja’s tool. The relative abundances of the main BA.2.86 lineages (grouped into JN.*, KP.*, LB.1, and other BA.2.86) are shown only for wastewater samples showing the presence of Omicron BA.2.86*.

Figure 4

Monthly characterization of circulating BA.2.86 lineages, as assessed by wastewater analysis: Data obtained by amplicon next-generation sequencing and Freyja analyses of wastewater samples collected between 18 October 2023 (date of first appearance of Omicron BA.2.86) and 1 July 2024 from the wastewater treatment plant of Pian della Genna (Perugia, Italy) are reported, aggregated by month, averaging the relative abundances of the main BA.2.86 lineages detected, namely, JN.1, JN.2, JN.3, KP.1, KP.2, KP.2.3, KP.3, KP.4, KP.5, and LB.1 (lineages JN.4 to JN.19 and all other BA.2.86 lineages are grouped into Other JN.* and Other BA.2.86, respectively).

Figure 5

Phylogenetic tree of SARS-CoV-2 whole-genome sequences retrieved from wastewater samples representative of the evolution of the virus during the study period. Phylogenetic analysis was performed on wastewater samples collected on 9 August 2023, 3 January 2024, and 29 May 2024, and based on the maximum likelihood method applied to the whole-genome consensus sequences obtained (coded as Italy_PG-INMI_2023-08-09, Italy_PG-INMI_2024-01-03, and Italy_PG-INMI_2024-05-29, respectively, and reported in red in the phylogenetic tree), as well as other sequences retrieved from wastewater-based studies carried out during the same period in Italy and Europe (reported in black). Nodes supported with bootstrap values ≥80 are marked with black dots, while Pango lineages obtained by Nextclade (clades.nextstrain.org, accessed on 21 May 2025) are shown alongside the tree.

Figure 6

Timeline of BA.2.86 lineages’ identification by clinical genomic surveillance and by wastewater analysis: The period of the first detection of each BA.2.86 lineage (namely. JN.1 to JN.3, KP.1 to KP.5, LB.1. and other BA.2.86) is represented by the colored dots placed in the green upper panel and in the lower yellow panel for clinical and wastewater samples, respectively.