Optimizing sample collection and data interpretation for effective wastewater-based epidemiology in combined sewer systems

Abstract

COVID-19 has spurred growth in the science surrounding wastewater-based epidemiology (WBE) pertaining to the detection of severe acute respiratory virus 2 (SARS-CoV-2) in waste streams as an early warning signal for public health. However, the highly variable wastewater environment has made it difficult to standardize an approach for sampling and analysis, especially in locations using combined sewer infrastructure. This study addresses knowledge gaps of WBE via three specific aims: (1) to compare diurnal fluctuations of SARS-CoV-2 and the human fecal indicator, pepper mild mottle virus (PMMoV) in wastewater treatment plant (WWTP) influent samples collected during dry versus wet weather conditions; (2) to assess accuracy of grab versus 24-hour composite samples collected under variable flow conditions; and (3) to examine changes in wastewater influent composition associated with rainfall derived inflow and infiltration (RDII) and impacts to SARS-CoV-2 and PMMoV abundance. Using droplet digital polymerase chain reaction (ddPCR), both SARS-CoV-2 and PMMoV were quantified hourly at two WWTPs in West Virginia during three wet and three dry weather events. A parallel configuration of two automated samplers was deployed at each WWTP to collect: (a) 24 grab samples, collected hourly, and (b) an equivalent 24-hour composite. Wastewater physiochemistry metadata (chemical oxygen demand, ammonia, conductivity, total suspended solids, turbidity, pH, temperature, and influent flow) was also collected. Results provided evidence of the influence of site-specific factors on viral abundance, including the potential role of septage haulers in skewed viral RNA abundance and RDII as a driver of overall viral concentrations. Significantly lower concentrations of SARS-CoV-2 were observed during wet weather days at both WWTPs (Mann Whitney U, p<0.001). On dry days, composite concentrations of SARS-CoV-2 ranged from 81% less than to 190% greater than the median hourly concentration, while results from wet weather days were more variable, ranging from 29% less than to 1,372% greater than the median hourly concentration, indicating potential flushing of viral RNA and dilution of grab samples to a point of near non-detection. Several physiochemical parameters showed significant positive correlations to viral RNA concentration regardless of dry or wet weather (Spearman’s rank, p<0.01), but consistency of observations was not preserved between both WWTPs, suggesting site-specific factors such as infiltration rate and community infection prevalence play a crucial role in determining viral presence and abundance in WBE samples. Collectively, this study identifies crucial determinants for WBE sample collection and data validation within combined sewer systems to enhance accuracy in reporting, inform local public health intervention, and mitigate the spread of infectious disease across communities.