Clinical Testing Underestimates COVID-19 Incidence and Variant Prevalence: Insights from WBE in Southern Nevada

Introduction. COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed approximately 4.5 million lives worldwide and continues to have severe impacts on social, economic, and educational aspects of life. Rapid vaccine rollouts were expected to mitigate the impacts of the pandemic and hopefully lead to herd immunity. However, heightened vaccine hesitancy coupled with the elimination of some public health measures and the emergence of the Delta variant hindered those outcomes and led to a dramatic spring/summer surge in the United States. Although clinical testing captured a large portion of that surge, there are indications that infections were more widespread than indicated by the confirmed case counts. This suggests clinical data may become less reliable as the pandemic progresses, particularly as fewer people seek reportable tests, and this highlights an opportunity for wastewater-based epidemiology (WBE). Although primarily known as a respiratory virus, SARS-CoV-2 causes secondary infections in the gastrointestinal (GI) tract of infected individuals. The viral genetic material is then shed in feces and can ultimately be detected in sewage. This presents an opportunity to obtain unbiased information on COVID-19 incidence because sewage-based data reflect infections from both symptomatic and asymptomatic individuals and do not require people to seek clinical testing. This process, known as WBE, has increased in popularity throughout the COVID-19 pandemic and has provided valuable public health information worldwide. In Southern Nevada, WBE commenced in March 2020 and ultimately expanded to include all of the major wastewater treatment facilities in the region, thereby providing critical public health surveillance for 2.3 million people in Las Vegas, North Las Vegas, Henderson, Boulder City, and unincorporated Clark County. This paper describes the outcomes from that effort, including (1) a comparison of trends for clinical and wastewater data, (2) an explanation of how WBE has been used to estimate infection totals for the region, and (3) data to demonstrate how sequencing has been used to identify variants of concern (VOCs) circulating in the community. Methods. All wastewater samples were concentrated either through hollow fiber ultrafiltration (HFUF) (REXEED 25S) or centrifugal ultrafiltration (Centricon Plus-70). Samples were spiked with bovine coronavirus as a recovery control prior to concentration and then extracted using a PureLink Viral RNA/DNA mini kit. SARS-CoV-2 genetic material was quantified using RT-qPCR by targeting four regions in the viral genome: N1, N2, E_Sarbeco, and orf1a. Recovery-corrected concentrations were averaged across the four gene targets and then correlated with daily zip-code-level clinical data obtained from the Southern Nevada Health District. A fecal shedding model was adapted from published literature and then calibrated to Nevada seroprevalence data. The final model was used to calculate predicted wastewater concentrations based on confirmed case counts and develop estimates for sewershed-specific and regional infection totals. Amplicon-based sequencing was performed with a CleanPlex SARS-CoV-2 Flex Panel from Paragon Genomics, and libraries were sequenced using an Illumina NextSeq 500. Variants of concern (VOCs) were successfully identified by searching for a minimum number of signature mutations/deletions. Results & Discussion. In Southern Nevada, there have been five COVID-19 case surges of varying severity since the onset of the pandemic, and wastewater concentrations have been strongly correlated with each of those surges (Figure 1). COVID-19 dynamics have been influenced by a number of competing factors, including discontinuation of stay-at-home orders, lifting/reinstitution of mask mandates, social/behavioral changes, and emergence of VOCs. For example, mutations associated with the alpha and epsilon variants were frequently detected in wastewater in the winter of 2020/2021, when concentrations initially peaked at Facility 1 at 8x105 gc/L (Figure 2). With the rise of the Delta variant in the summer of 2021, Facility 1 concentrations hit a new all-time high of 9x105 gc/L (Figure 2). These wastewater surveillance data also highlight a critical drawback of clinical testing, namely that clinical testing becomes unreliable when people no longer seek those tests. During the winter surge, COVID-19 incidence peaked in the Facility 1 service area at approximately 86 new daily cases per 100,000 people. However, with even higher wastewater concentrations, COVID-19 incidence during the summer surge peaked at only 42 new daily cases per 100,000 people. A similar discrepancy was observed across all of Southern Nevada (Figure 3). This is presumably due to a combination of factors, including reductions in random and mandated testing, increased incidence of asymptomatic or mild infections (e.g., due to vaccination or infections among lower-risk demographics), and increased availability of non-reportable at-home tests. Hence, clinical testing may significantly underestimate actual disease incidence and prevalence, particularly as the pandemic progresses. As of August 2021, WBE suggests that 1.2 million people in Southern Nevada (out of a total population of 2.2 million) have been infected since the start of the pandemic, with clinical testing underestimating the total number of infections by 4.2-fold (Table 1). Looking to the future, WBE has the potential to fill critical data gaps and provide a more comprehensive window into community health conditions for both pathogens and chemicals of interest (e.g., illicit drugs).