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Wastewater testing for SARS-CoV-2 provided useful public health information during the COVID-19 pandemic yet was underutilized in rural communities. We addressed this gap by implementing wastewater surveillance and assessing its performance in 10 communities in Eastern Kentucky. We collected wastewater samples 1–2 times weekly at 10 wastewater treatment plants (WWTPs) from May 2021 until April 2023 and measured SARS-CoV-2 RNA concentrations using polymerase chain reaction testing. We calculated time-lagged correlations between wastewater concentrations and county-level reported COVID-19 cases by site. We developed a generalized linear model to estimate COVID-19 incidence from wastewater SARS-CoV-2 concentrations. The 10 participating WWTPs served 2430 to 35,575 customers, and 90% were in rural counties. We cumulatively analyzed 818 wastewater samples. Correlations between wastewater SARS-CoV-2 concentrations and COVID-19 cases were significant at seven of the WWTPs and were strongest during the Delta variant period. The incidence density model predicted more COVID-19 cases during the latter study period (May 2022–April 2023) than were officially reported. Wastewater surveillance data in these rural communities corroborated clinical case data and may have more accurately described community disease levels later in the pandemic. 

Wastewater-based epidemiology (WBE) measures pathogens in wastewater to monitor infectious disease prev- alence in communities. Due to the high dilution of pathogens in sewage, a concentration method is often required to achieve reliable biomarker signals. However, most of the current concentration methods rely on expensive equipment and labor-intensive processes, which limits the application of WBE in low-resource settings. Here, we compared the performance of four inexpensive and simple concentration methods to detect SARS-CoV-2 in wastewater samples: Solid Fraction, Porcine Gastric Mucin-conjugated Magnetic Beads, Calcium Flocculation- Citrate Dissolution (CFCD), and Nanotrap® Magnetic Beads (NMBs). The NMBs and CFCD methods yielded the highest concentration performance for SARS-CoV-2 (~16-fold concentration and ~ 41 % recovery) and require <45 min processing time. CFCD has a relatively low consumable cost (<$2 per four sample replicates). All methods can be performed with basic laboratory equipment and minimal electricity usage which enables further application of WBE in remote areas and low resource settings.