The World Health Organization (WHO) classified COVID-19 as a global pandemic, with the situation ultimately requiring unprecedented measures to mitigate the effects on public health and the global economy. Although SARS-CoV-2 (the virus responsible for COVID-19) is primarily respiratory in nature, multiple studies confirmed its genetic material could be detected in the feces of infected individuals, thereby highlighting sewage as a potential indicator of community incidence or prevalence. Numerous wastewater surveillance studies subsequently confirmed detection of SARS-CoV-2 RNA in wastewater and wastewater-associated solids/sludge. However, the methods employed in early studies vary widely so it is unclear whether differences in reported concentrations reflect true differences in epidemiological conditions, or are instead driven by methodological artifacts. The current study aimed to compare the performance of virus recovery and detection methods, detect and quantify SARS-CoV-2 genetic material in two Southern Nevada sewersheds from March–May 2020, and better understand the potential link between COVID-19 incidence/prevalence and wastewater concentrations of SARS-CoV-2 RNA. SARS-CoV-2 surrogate recovery (0.34%–55%) and equivalent sample volume (0.1 mL–1 L) differed between methods and target water matrices, ultimately impacting method sensitivity and reported concentrations. Composite sampling of influent and primary effluent resulted in a ∼10-fold increase in concentration relative to corresponding grab primary effluent samples, presumably highlighting diurnal variability in SARS-CoV-2 signal. Detection and quantification of four SARS-CoV-2 genetic markers (up to ∼10 6 gene copies per liter), along with ratios of SARS-CoV-2 to pepper mild mottle virus (PMMoV), exhibited comparability with public health data for two sewersheds in an early phase of the pandemic. Finally, a wastewater model informed by fecal shedding rates highlighted the potential significance of new cases (i.e., incidence rather than prevalence) when interpreting wastewater surveillance data.

a Drinking water filters may be operated to promote or deter biological activity through upstream oxidant addition. While there are several water quality benefits from biofiltration, microbial growth in biofilters warrants further investigation. In this study, routine monitoring detected target DNA sequences for Naegleria fowleri in source water and Acanthamoeba spp. in source water and biofilter effluent, triggering further microbial community characterization. Full-scale anthracite and granular activated carbon (GAC) filters receiving chlorinated waters were compared in terms of effluent water quality (i.e., turbidity and particle counts), biological activity (i.e., adenosine triphosphate (ATP)), and the composition of the microbial community (i.e., 16S/18S rRNA gene sequencing, free-living amoeba). Because of rapid chlorine quenching by GAC, greater biomass development was observed in the GAC biofilter (ATP = 5 × 10 3 -5 × 10 4 pg cm −3 media) than the anthracite filter (ATP = 4 × 10 2 -1 × 10 3 pg cm −3 media). Due to possible sloughed biomass, GAC effluent also had consistently greater turbidity, particle counts, and cellular ATP than the anthracite filter. 16S rRNA gene sequencing revealed distinct taxonomic differences between the anthracite and GAC filters, and GAC also hosted a more diverse population (Shannon index: GAC = 3.7-5.0 and anthracite = 2.4-2.9). At the genus level, the anthracite filter contained mostly Undibacterium-like taxon (45-68%), while the GAC biofilter was dominated by Massilia (8-36%), Herbaspirillum (2-44%), and unknown Comamonadaceae (9-18%), among others. The presence of viable free-living amoebas was also detected in the GAC biofilter media. Further characterization of the eukaryotic 18S rRNA gene showed that anthracite predominantly harbored copepod Leptodiaptomus (67%), while a majority of the sequences in GAC are unknown.