Long-term biological surveillance of SARS-CoV-2 in critical points for municipal sewage catchment in light of wastewater-based epidemiology, public health and environmental hygiene.

Routine monitoring and wastewater-based bio-surveillance represents a strong supporting tool for following and understanding SARS-CoV-2 endemicity or epidemic dynamics in society. The main objective of the research was a two-year qualitative and quantitative monitoring program of SARS-CoV-2 in sewage in a model urban agglomeration (the capital city), including the development of an algorithm for action in this area and a joint evaluation of procedures with a water and sewage company (public administration sector). To resolve problems, we continuously investigated inflows and outflows of four wastewater treatment plants and raw sewage of the whole catchment (including healthcare units, airport and military areas) within the Warsaw capital city for SARS-CoV-2, during two waves of COVID-19 in 2023 and 2024. The screening was performed by RT-qPCR (N1, N2, E probes) and NGS metagenomics analysis. The average cumulative load of the virus in sewage for the whole tested period was 3.5 × 105 genomic copies per litre. The highest load was found for wastewater inflows and the lowest one was found for housing estates and wastewater outflows. Higher viral concentrations in the wastewater than expected in medical records suggested a higher number of undiagnosed COVID-19 infections within the community. The obtained genotypes segregated within 11 clades (21I to 24 F), making '23 and '24 waves distinguishable. We consider our results and methodological approaches hold significant potential for public health, with particular emphasis on the needs of the Directive (EU) 2024/3019 of the European Parliament and of the Council.