Wastewater surveillance emerged as a critical public health tool during the COVID-19 pandemic, enabling early detection of community-level pathogen circulation independent of clinical testing. Its ability to capture signals from both symptomatic and asymptomatic individuals highlighted the importance of optimizing sampling methodologies to improve sensitivity and reliability. A key question is whether the several-fold increase in SARS-CoV-2 detectability observed when using passive tampon swab sampling compared with paired grab samples also applies to other respiratory viruses, including influenza A (including its avian influenza H5N1 subtype), influenza B, and respiratory syncytial virus (RSV). We collected 24 h passive swab samples with same-day grab samples from Detroit sewersheds, concentrated and purified nucleic acids, and using RT-ddPCR, quantified respiratory syncytial virus, SARS-CoV-2, influenza A, influenza B, and H5N1 influenza A viruses using markers RSV, SC2, InfA, InfB, and H5, respectively. Samples testing positive for H5 (marker for H5N1 influenza A) were further analyzed by targeted PCR and amplicon sequencing. Across three sites, median 24 h swab:grab ratios of virus copies were 7.0 for RSV, 9.2 for SC2, 9.9 for InfA, and 3.6 for InfB. A 239 bp hemagglutinin sequence from a sample with a strong H5 signal (795 copies/10 mL) had 100% identity to avian influenza viruses from Canada geese. Twenty-four-hour swab sampling greatly improves viral detectability across diverse targets and enabled the first confirmed detection of H5 in Detroit wastewater. Combined with magnetic bead purification, the overall sensitivity gain over conventional PEG-NaCl-Qiagen methods is approximately 36-fold, enabling earlier warning of community pathogens than grab samples. By integrating 24 hour passive swab sampling with high-efficiency nucleic acid purification, we expand the sensitivity of wastewater surveillance to enable detection and confirmation of low-abundance pathogens like avian influenza (H5).
Ghosh et al. (Thu,) studied this question.