High-Frequency, High-Throughput Quantification of SARS-CoV-2 RNA in Wastewater Settled Solids at Eight Publicly Owned Treatment Works in Northern California Shows Strong Association with COVID-19 Incidence

A number of recent retrospective studies have demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater are associated with coronavirus disease 2019 (COVID-19) cases in the corresponding sewersheds. Implementing high-resolution, prospective efforts across multiple plants depends on sensitive measurements that are representative of COVID-19 cases, scalable for high-throughput analysis, and comparable across laboratories. We conducted a prospective study across eight publicly owned treatment works (POTWs). A focus on SARS-CoV-2 RNA in solids enabled us to scale up our measurements with a commercial lab partner. Samples were collected daily, and results were posted to a website within 24 h. SARS-CoV-2 RNA in daily samples correlated with the incidence of COVID-19 cases in the sewersheds; a 1 log₁₀ increase in SARS-CoV-2 RNA in settled solids corresponds to a 0.58 log₁₀ (4×) increase in sewershed incidence rate. SARS-CoV-2 RNA signals measured with the commercial laboratory partner were comparable across plants and comparable to measurements conducted in a university laboratory when normalized by pepper mild mottle virus (PMMoV) RNA. Results suggest that SARS-CoV-2 RNA should be detectable in settled solids for COVID-19 incidence rates of >1/100,000 (range, 0.8 to 2.3 cases per 100,000). These sensitive, representative, scalable, and comparable methods will be valuable for future efforts to scale up wastewater-based epidemiology. <b>IMPORTANCE</b> Access to reliable, rapid monitoring data is critical to guide response to an infectious disease outbreak. For pathogens that are shed in feces or urine, monitoring wastewater can provide a cost-effective snapshot of transmission in an entire community via a single sample. In order for a method to be useful for ongoing COVID-19 monitoring, it should be sensitive for detection of low concentrations of SARS-CoV-2, representative of incidence rates in the community, scalable to generate data quickly, and comparable across laboratories. This paper presents a method utilizing wastewater solids to meet these goals, producing measurements of SARS-CoV-2 RNA strongly associated with COVID-19 cases in the sewershed of a publicly owned treatment work. Results, provided within 24 h, can be used to detect incidence rates as low as approximately 1/100,000 cases and can be normalized for comparison across locations generating data using different methods.