Abstract / Summary
ABSTRACT Hospitals generate wastewater enriched with human-derived microbes, making them strong sites for focused, near-source surveillance of pathogens. We compared in-sewer 24-h composite and passive sampling at two hospital discharge locations and influent sampling at a municipal wastewater treatment plant (WWTP). We also evaluated three extraction workflows to determine how sampling and extraction methods influence detection sensitivity across faecal indicators (HF183, crAssphage) and clinically relevant pathogens (SARS-CoV-2, human adenovirus, Campylobacter jejuni). Across 14 consecutive days, HF183 and crAssphage quantitative polymerase chain reaction (qPCR) cycle threshold (Ct) signals were stronger in hospital wastewater than at the WWTP, consistent with greater human faecal input at the near-source sites. crAssphage and human adenovirus were detected with comparable Ct values in passive and liquid samples, whereas the bacterial targets HF183 and C. jejuni were detected less sensitively by passive sampling, particularly at one hospital site. This pattern was consistent with site-specific wastewater composition and flow dynamics affecting membrane retention. SARS-CoV-2 variant sequencing from hospital wastewater resolved five lineages, with the emerging XEC recombinant identified up to 14 days before its detection in municipal wastewater or clinical sequencing. Overall, sampling method, location, and analytical workflow all influenced detection sensitivity, highlighting the need for context-specific method optimisation in near-source wastewater surveillance.