When coronavirus disease 2019 (COVID-19) was declared a public health emergency by the World Health Organization in 2020, public health laboratories in the United States (U.S.) played a pivotal role in determining case infection levels. However, these laboratories were soon overwhelmed by surveillance activities that were required to quantify and track emerging SARS-CoV-2 variants. In response to this challenge, the U.S. Centers for Disease Control and Prevention (CDC) initiated a National Wastewater Surveillance System (NWSS) to complement more traditional public health surveillance efforts. (1) SARS-CoV-2 viral RNA can be found in human feces and urine, enabling investigators to monitor wastewater and provide estimates of COVID-19 incidence or community prevalence. (2) Since March 2020, we and others have demonstrated that wastewater surveillance can provide an early detection system for high-priority pathogens, antimicrobial resistance markers, high risk substances, and emerging SARS-CoV-2 variants. (3−15)

In addition to reporting on SARS-CoV-2 viral RNA in wastewater, several studies have described the detection of viral material and human fecal contamination in flood control infrastructure due to overflowing sanitary sewers, sewer collection system leaks, and direct human inputs. (16−18) In cities like Las Vegas, environmental factors (e.g., relatively mild winters) coupled with city ordinances criminalizing unsheltered homelessness have led to the establishment of encampments in remote areas, including flood control channels, where makeshift toilets and open defecation are common. (18) These unsheltered individuals are at higher risk of infection, morbidity, and disease-associated mortality because of their limited access to health care services. (19−21) Thus, wastewater surveillance of homeless shelters (8) and environmental surveillance of flood control infrastructure may help characterize public health conditions and disease transmission, including for COVID-19, within this particularly vulnerable population that is often underrepresented in traditional clinical surveillance data.

SARS-CoV-2 viral RNA can persist for up to several weeks in environmental waters, and potentially longer depending on the temperature of the water. (22) In addition, SARS-CoV-2 infected individuals shed maximum viral RNA in fecal matter at the onset of infection and up to seven months after the initial infection. (8,22−24) While previous studies on SARS-CoV-2 in surface waters, including stormwater, have documented SARS-CoV-2 RNA levels by quantitative polymerase chain reaction (qPCR), (16,17) whole genome sequencing (WGS) of samples from flood control channels (e.g., urban runoff and stormwater) to generate SARS-CoV-2 variant information is less common. (16,17,25) This could be due to challenges with collection and analysis of these complex samples, specifically performing WGS on low viral loads or in the presence of unique PCR inhibitors which negatively influence the success of sequencing workflows. (7,26) Obtaining knowledge about the SARS-CoV-2 variant composition in urban runoff and stormwater could support public health surveillance of new reservoirs of viral variants. Therefore, the objectives of this study were to (1) determine if SARS-CoV-2 RNA could be detected in environmental water samples collected from flood control infrastructure that is known to be impacted by unsheltered individuals, (2) conduct amplicon-based WGS of SARS-CoV-2 from these environmental water samples, (3) compare SARS-CoV-2 variants present in the environmental water samples with those circulating in the local community (via wastewater and clinical surveillance), and (4) investigate if novel mutations could be detected...