INTEGRATED APPROACHES IN WASTEWATER SURVEILLANCE OF SARS-COV-2: MONITORING, PERSISTENCE, SAMPLING OPTIMIZATION, AND IMPACTS ON MICROBIAL COMMUNITY STRUCTURE

Wastewater-based epidemiology (WBE) serves as a crucial tool for monitoring SARS-CoV-2 prevalence on university campuses, yet concerns persist regarding its effectiveness as an early warning system for COVID-19 outbreaks. This study aimed to address these concerns through a comprehensive field trial conducted at the University of Tennessee, Knoxville. We investigated correlations between SARS-CoV-2 concentrations, both with and without normalization using Pepper Mild Mottle Virus (PMMoV), and various parameters. Significant positive correlations were observed between SARS-CoV-2 concentrations and active cases in the weeks prior to, during, and following the monitoring period, unaffected by PMMoV normalization. Concurrently, the persistence of SARS-CoV-2 RNA in raw sewage, akin to municipal wastewater, was examined. Temperature emerged as a significant factor influencing decay rate constants of SARS-CoV-2 RNA, with higher temperatures accelerating decay. Notably, differences in decay rates were observed between SARS-CoV-2 RNA and non-enveloped PMMoV RNA, underscoring the sensitivity of SARS-CoV-2 RNA to temperature variations. Moreover, inadequate sampling approaches in wastewater analysis can introduce biases and inaccuracies. To mitigate this, various composite sampling modes were employed in University of Tennessee residence halls. Results demonstrated that PMMoV RNA exhibited higher abundance and lower variability compared to SARS-CoV-2 RNA. A 6-hour morning window for composite sampling emerged as a pragmatic and cost-effective strategy for generating representative samples of pathogenic SARS-CoV-2 RNA. Lastly, microbial dynamics in sewage during the COVID-19 pandemic were investigated using 16S rRNA sequencing. Positive samples for SARS-CoV-2 displayed reduced bacterial diversity and distinct microbial compositions compared to negative samples. The identified correlation between the enteric pathogen rate and the percentage of potential pathogens at sampling sites adds a noteworthy dimension to our understanding of COVID-19, particularly in the significant association between the enteric pathogen rate and the pathogen rate in positive SARS-CoV-2 samples. Furthermore, the significant correlation in the potential pathogen rate between positive and negative SARS-CoV-2 sewage samples may be linked to the enduring effects of microbial dysbiosis observed during COVID-19 recovery. Collectively, these findings offer valuable insights into the application, limitations, and implications of WBE for COVID-19 surveillance on university campuses.