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  1. 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. 
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    Free, publicly-accessible full text available March 1, 2027
  2. Wastewater-based epidemiology (WBE) played a vital role during the COVID-19 pandemic by providing early warnings of outbreaks through SARS-CoV-2 RNA detection in sewage. Many rural communities did not benefit from WBE because limited centralized sewer infrastructure challenged conventional WBE surveillance strategies. We present a multi-agent computer model simulating COVID-19 spread in a U.S. county with both sewered and non-sewered zones to assess the performance of WBE in this setting. We evaluate how the sewage service status of the first SARS-CoV-2 carrier, cross-zone community mobility, and WBE detection thresholds influence outbreak detection timing at the county’s wastewater treatment plant under basic reproduction numbers (R0) of 4 and 8. Our key findings include that (1) a detection threshold of 10 gc/mL can identify outbreaks up to six days earlier than a threshold of 50 gc/mL; (2) outbreaks originating in non-sewered zones are detected 1–2 days later, compared with outbreaks in sewered zones; and (3) cross-zone community mobility impacts detection timing only when outbreaks begin in non-sewered zones. Furthermore, once detected, disease prevalence can increase by five- to eleven-fold within the following week. These results underscore the importance of WBE sensitivity and tailored surveillance strategies in both sewered and non-sewered zones of a community. Strengthening WBE capabilities at local treatment facilities can improve early outbreak detection, thereby supporting timely public health interventions. 
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    Free, publicly-accessible full text available December 1, 2026
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