Abstract 1269 Effect of sample collection variables on viral RNA recovery from manholes for wastewater-based epidemiology

Wastewater is a valuable tool for tracking infectious agents, such as SARS-CoV-2, to enable a robust public health response to incipient epidemics. Although wastewater viral signal is broadly correlated with infection level, thus far, WBE has failed to accurately predict numbers of infections within a sewage catchment basin. One reason may be that sampling variables are not strictly controlled. Our objective was to investigate sampling variables that influence the recovery of viral RNA from wastewater using a ubiquitous human fecal marker, Pepper Mild Mottle Virus (PMMoV). For sixteen weeks, we sampled three different strata of wastewater in a single manhole along with temperature and pH. Viral RNA was consistently detected in these three layers by RT-qPCR, with the highest signal in the sediment layer. This layer also had the lowest temperature and pH, suggesting these may influence viral persistence. Despite the high concentration of viral RNA in the sediment layer, PCR-based methods suffered the most inhibition in this layer. In conclusion, we find that inadvertent variability in sampling depth could dramatically affect the recovery of viral RNA signal from wastewater, potentially because of changing physical and chemical properties. Our work demonstrates the need for minimizing sampling variability in WBE. Acknowledging MDHHS, and Kent County Health Department, MI for funding this research project.