Comparative Assessment of Wastewater-Based Surveillance Normalization Methods to Improve Pathogen Monitoring in Rural Sewersheds

Many in-sewer dynamics that can impact the fidelity of wastewater-based surveillance results remain understudied. Some conventional approaches for normalizing pathogen signals in sewersheds may not be appropriate when there is substantial inflow and infiltration (I&I). Our objective for this study was to evaluate the effect of multiple normalization approaches on wastewater pathogen signals at the WWTP influent and across a small rural sewershed (<3000 people) with different levels of I&I. We collected wastewater samples monthly, from 2022 to 2023, from the wastewater treatment plant (WWTP) influent and 11 additional sewer system nodes with well-characterized I&I impacts. We quantified concentrations of SARS-CoV-2, norovirus GII, and rotavirus at the WWTP influent and subsewershed sites, and compared normalization approaches using flow, population, physicochemical parameters (COD, TSS, NH₃(aq), PO₄₃^--P), and human fecal markers (crAssphage, HF183, mtDNA). Overall, our findings suggest that in systems with substantial I&I, some commonly used normalization approaches, particularly those using physicochemical parameters, may inadvertently introduce additional variability in viral signals measured at WWTP influent and result in wastewater measures that are more closely associated with precipitation trends rather than with pathogen signals. Normalization with human fecal markers appears to be a relatively robust option for sewersheds impacted by substantial I&I.