Tracking viral RNA loads during in-sewer transport: insights from real-world field experiments.

Wastewater-based surveillance (WBS) is widely used to monitor respiratory viruses, yet uncertainties remain regarding how viral RNA concentrations in wastewater reflect infection dynamics. Specifically, diurnal variation in shedding and RNA losses during in-sewer transport can impact measured signals. We conducted a field study in a 5-km trunk sewer (travel time of one hour), with sampling campaigns in November 2024 and January 2025. Wastewater was sampled at the sewer inlet and outlet using autosamplers collecting time-proportional one-hour composite samples over 24 h. The one-hour composite samples were analyzed for assessing diurnal variations, and 24-hour composites for signal change. Biofilms from the sewer-pipe walls were collected at three locations. Nucleic acids were extracted, and SARS-CoV-2, Influenza A/B, and Respiratory Syncytial Virus (RSV) RNA were quantified using a multiplex digital PCR assay. In the January 2025 campaign, viruses showed pronounced diurnal variations, with morning load peaks. Moreover, SARS-CoV-2 and Influenza B RNA in the bulk liquid decreased during in-sewer transport, with modelled reductions of 43 % and 44 %; changes in the other targets were uncertain, with credible intervals spanning zero. In the sewer system investigated, biofilms seem to be a minor reservoir for viral RNA; additionally, for SARS-CoV-2, sequencing did not reveal a different biofilm-associated population. Our study provides a full-scale assessment of in-sewer transport effects on viral RNA and highlights the need to account for complex in-sewer dynamics when interpreting WBS data.