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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.

  • Wolfe, Marlene K1
  • Topol, Aaron2
  • Knudson, Alisha2
  • Simpson, Adrian2
  • White, Bradley2
  • Vugia, Duc J3
  • Yu, Alexander T3
  • Li, Linlin4
  • Balliet, Michael5
  • Stoddard, Pamela4
  • Han, George S4
  • Wigginton, Krista R6
  • Boehm, Alexandria B1
  • 1 Department of Civil & Environmental Engineering, Stanford Universitygrid.168010.e, Stanford, California, USA.
  • 2 Verily Life Sciences, South San Francisco, California, USA.
  • 3 California Department of Public Health, Infectious Diseases Branch, Richmond, California, USA.
  • 4 County of Santa Clara Public Health Department, San Jose, California, USA.
  • 5 County of Santa Clara Department of Environmental Health, San Jose, California, USA.
  • 6 Department of Civil & Environmental Engineering, University of Michigan, Ann Arbor, Michigan, USA.
Published Article
Publication Date
Oct 26, 2021
DOI: 10.1128/mSystems.00829-21
PMID: 34519528


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. IMPORTANCE 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.

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