Solid Evidence and Liquid Gold: Trade-Offs of Processing Settled Solids, Whole Influent, or Centrifuged Influent for Codetecting 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 the 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 11 facilities at three time points. We demonstrate that one workflow can be used to simultaneously detect all of the targets. Comparing normalization of targets by concentration of PMMoV, carjivirus, and 16S rRNA, we show that using different controls together can complicate the interpretation of concentrations across targets. We observed similar detection rates and nucleic acid concentrations for the targets examined in this study across all three sample types with slightly higher concentrations in centrifuged influent. These results suggest that centrifuged influent is a viable option and can circumvent the limitation of varying residence times for primary settled solids.