Estimating Relative Abundance of 2 SARS-CoV-2 Variants Through Wastewater Surveillance at 2 Large Metropolitan Sites, United States

Alexander T. Yu; Bridgette Hughes; Marlene K. Wolfe; Tomas Leon; Dorothea Duong; Angela Rabe; Lauren C. Kennedy; Sindhu Ravuri; Bradley J. White; Krista R. Wigginton; Alexandria B. Boehm; Duc J. Vugia


Emerging Infectious Diseases. 2022;28(5):940-947. 

In This Article

Abstract and Introduction


Monitoring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) is critical for public health management of coronavirus disease. Sequencing is resource-intensive and incompletely representative, and not all isolates can be sequenced. Because wastewater SARS-CoV-2 RNA concentrations correlate with coronavirus disease incidence in sewersheds, tracking VOCs through wastewater is appealing. We developed digital reverse transcription PCRs to monitor abundance of select mutations in Alpha and Delta VOCs in wastewater settled solids, applied these to July 2020–August 2021 samples from 2 large US metropolitan sewersheds, and compared results to estimates of VOC abundance from case isolate sequencing. Wastewater measurements tracked closely with case isolate estimates (Alpha, rp 0.82–0.88; Delta, rp 0.97). Mutations were detected in wastewater even at levels <5% of total SARS-CoV-2 RNA and in samples available 1–3 weeks before case isolate results. Wastewater variant monitoring should be strategically deployed to complement case isolate sequencing.


By November 2021, the coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had claimed >5 million lives worldwide, including >700,000 in the United States.[1–3] Since its emergence in late 2019, SARS-CoV-2 has mutated, resulting in some variants categorized by the World Health Organization as variants of concern (VOCs). VOCs have evidence of potential increased infectiousness, immune evasion, and clinical severity, and they have spread globally. Some VOCs, such as Alpha and Delta, have become the predominant strain at different times and regions.[4,5] COVID-19 diagnostics, therapeutics, or vaccines may have decreased effectiveness against VOCs.[6,7] As of November 2021, VOCs in the United States included the Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) variants.[3]

Monitoring for VOCs is critical for management of the ongoing COVID-19 pandemic, enabling public health officials to track their public health impact, implement control measures, and allocate resources effectively. Detection of SARS-CoV-2 variants occurs primarily through genomic sequencing of isolates collected for PCR-based diagnosis of persons with active COVID-19 infection. Sequencing is resource- and time-intensive and has limits on capacity because of equipment, reagents, and trained personnel.[8] As such, complete and timely sequencing of case isolates is not feasible or practical, particularly when case numbers have been high. During January 2020–September 2021, <3% of COVID-19 cases in the United States had isolates that were sequenced and available on public repositories.[3] Nonrandom selection of isolates for sequencing and nonuniform result reporting could make results susceptible to bias and not truly representative of circulating variants.[4,8,9] Also, substantial delays can occur between isolate collection, sequencing and availability of results to public health.[9] Given its timeliness, representativeness, and comparatively low costs, wastewater surveillance for VOCs can be a useful supplement to case-based sequencing surveillance.[10–12]

Since early in the pandemic, wastewater samples have been collected and analyzed to quantify the amount of SARS-CoV-2 RNA in sewage. Estimates of viral RNA abundance in sewage correlate closely with reported COVID-19 case counts for the catchment area (sewershed)[13,14] and provide a comprehensive snapshot of real-time community transmission independent of individual care-seeking or testing behavior. Therefore, there is a strong interest in determining if wastewater can also provide useful information on circulating VOCs.[15] Both sequencing and PCR assays targeting specific mutations have been proposed as methods to detect mutations and deletions in SARS-CoV-2 RNA in wastewater.

Variant monitoring using environmental samples presents technical challenges. Variants are characterized by the presence of multiple mutations on the same RNA genome, and some share ≥1 mutations.[16] Unlike isolates from an individual case, which consist of a single genome, wastewater samples likely contain material from multiple SARS-CoV-2 variants shed from different persons, each variant at low concentrations and in various states of genomic integrity because of degradation.[17] Therefore, because wastewater contains a complex mixture of SARS-CoV-2 RNA fragments, the presence of ≥1 variant mutation sequences does not alone prove that the variant is present in wastewater.

We developed targeted digital reverse transcription PCR mutation assays to retrospectively and prospectively monitor wastewater settled solids for the presence and abundance of mutations present in the Alpha (B.1.1.7) and Delta (B.1.617.2) VOCs. We chose wastewater solids because they contain orders of magnitude higher concentrations of viral RNA than wastewater influent;[18,19] previous work has documented a strong coupling between SARS-CoV-2 RNA concentrations in wastewater solids and incidence in the associated population contributing to the wastewater.[19] We prospectively monitored wastewater solids of a large metropolitan sewershed in California (San Jose), USA, during July 2020–August 2021 for a deletion present in the Alpha variant. We then retrospectively measured the abundance of this deletion in a second large metropolitan area (Sacramento, CA, USA) where samples had been routinely collected. We also measured concentrations of mutations suggestive of Delta in both sewersheds. We then compared these totals against estimates of Alpha and Delta abundance in each of these sewersheds by using COVID-19 case isolate sequencing data available to the California Department of Public Health (CDPH).