Solid evidence and liquid gold: trade-offs of processing settled solids, whole influent, or centrifuged influent for co-detecting viral, bacterial, and eukaryotic pathogens in wastewater

Effective methods for simultaneously measuring viral, bacterial, protozoan, and fungal pathogens in wastewater are needed. Here, we investigate how sample type and nucleic acid extraction protocols affect broad-range pathogen detection. We compared methods for analyzing wastewater solids and whole influent by dPCR detection of spiked and endogenous targets including DNA and RNA viruses (mpox, norovirus, SARS-CoV-2), bacteria ( Clostridium difficile, Campylobacter jejuni ), protozoa ( Cryptosporidium spp .), fungi ( Candida auris ), and antibiotic resistance genes. Using selected methods, we then analyzed date-matched 1) solid, 2) centrifuged influent, and 3) whole influent samples collected from eleven facilities at three time points. We demonstrate that one workflow can be used to simultaneously detect all targets and that all sample types yielded similar detection levels and nucleic acid concentrations. Comparing normalization of targets by concentration of PMMoV, carjivirus, and 16S, we show that using different controls together can complicate interpretation of concentrations across targets. Centrifuged influent produced comparable or higher target concentrations overall, suggesting that centrifuged influent is a viable option when settled solids are not available and can circumvent the limitation of varying residence times for primary settled solids.