Estimating relative abundance of two SARS-CoV-2 variants through wastewater surveillance at two large metropolitan sites (preprint)

Monitoring SARS-CoV-2 variants of concern (VOCs) is critical for public health management of COVID-19. Case isolate sequencing is resource-intensive and not all isolates can be sequenced, and thus are not representative. However, since wastewater SARS-CoV-2 RNA concentrations correlate with COVID-19 case incidence in sewersheds, tracking VOCs through wastewater is appealing. We developed targeted digital RT-PCR assays to monitor abundance of select mutations in Alpha and Delta VOCs in wastewater settled solids, applied these to July 2020-August 2021 samples from two large metropolitan sewersheds, and compared results to estimates of variant abundance from case isolate sequencing. Wastewater measurements tracked closely with case isolate estimates at each site (rp= 0.82, 0.88 for Alpha and rp= 0.97 for Delta). Mutations were detected in wastewater measurements even at levels <5% of circulating virus and in samples available weeks before case isolate results. Therefore, wastewater variant monitoring should be strategically deployed to complement case isolate sequencing.

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 (preprint)

A number of recent retrospective studies have demonstrated that SARS-CoV-2 RNA concentrations in wastewater are associated with 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-hours. SARS-CoV-2 RNA in daily samples correlated to incidence COVID-19 cases in the sewersheds; a 1 log 10 increase in SARS-CoV-2 RNA in settled solids corresponds to a 0.58 log 10 (4X) increase in sewershed incidence rate. SARS-CoV-2 RNA signals measured with the commercial laboratory partner were comparable across plants and 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 > 1/100,000 (range 0.8 - 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 hrs, 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.