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Abstract

Wastewater-based testing for SARS-CoV-2 has been implemented widely to complement clinical testing as an innovative and effective approach to infectious disease surveillance during the COVID-19 pandemic. This approach to disease surveillance is referred to as wastewater-based epidemiology, or “WBE”, where wastewater is viewed as a pooled, collective, representative sample from the community. When monitored overtime, viral titers of SARS-CoV-2 in wastewater have been demonstrated to correlate with clinical cases of COVID-19, providing an early-warning of outbreaks when clinical testing is limited, underutilized, or replaced with at-home testing. When paired with molecular epidemiology, WBE can be utilized to monitor the abundance of known, circulating variants of SARS-CoV-2 and to detect novel mutations of emerging variants. As the utility of wastewater-based epidemiology expands, there is growing interest in expanding the capacity of surveillance efforts, locally, nationally, and internationally. In order to facilitate the adoption and implementation of wastewater surveillance, this dissertation research investigated tools for the cost-efficient and accurate monitoring of both viral titers and circulating variants of SARS-CoV-2 in wastewater. In this work, we described the application of a simplified workflow for routine wastewater surveillance, using direct column-based RNA extraction for recovery and quantification of SARS-CoV-2 from influent wastewater samples. Bypassing any sample concentration steps, this simplified workflow provides an accessible alternative to previously published methods that may be particularly useful in resource-constrained settings. While it has been common practice to normalize SARS-CoV-2 viral titers to human fecal indicators in wastewater, we demonstrate that the prevalence and shedding of fecal indicators in stool is highly variable across a population. Normalizing viral titers to these fecal indicators likely introduces more uncertainty than it resolves. Lastly, using constructed wastewater mock communities, we systematically assessed the efficacy of three tiled amplicon enrichment methods for whole genome sequencing of SARS-CoV-2 variants from wastewater. We found sequencing SASRS-CoV-2 from wastewater to be largely inefficient, though sequences can provide accurate estimates of variant abundance when libraries are sequenced deeply.

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