Reproducibility and sensitivity of 36 methods to quantify the SARS-CoV-2 genetic signal in raw wastewater: findings from an interlaboratory methods evaluation in the U.S.

In response to COVID-19, the international water community rapidly developed methods to quantify the SARS-CoV-2 genetic signal in untreated wastewater. Wastewater surveillance using such methods has the potential to complement clinical testing in assessing community health. This interlaboratory assessment evaluated the reproducibility and sensitivity of 36 standard operating procedures (SOPs), divided into eight method groups based on sample concentration approach and whether solids were removed. Two raw wastewater samples were collected in August 2020, amended with a matrix spike (betacoronavirus OC43), and distributed to 32 laboratories across the U.S. Replicate samples analyzed in accordance with the project's quality assurance plan showed high reproducibility across the 36 SOPs: 80% of the recovery-corrected results fell within a band of ±1.15 log10 genome copies per L with higher reproducibility observed within a single SOP (standard deviation of 0.13 log10). The inclusion of a solids removal step and the selection of a concentration method did not show a clear, systematic impact on the recovery-corrected results. Other methodological variations (e.g., pasteurization, primer set selection, and use of RT-qPCR or RT-dPCR platforms) generally resulted in small differences compared to other sources of variability. These findings suggest that a variety of methods are capable of producing reproducible results, though the same SOP or laboratory should be selected to track SARS-CoV-2 trends at a given facility. The methods showed a 7 log10 range of recovery efficiency and limit of detection highlighting the importance of recovery correction and the need to consider method sensitivity when selecting methods for wastewater surveillance.

Correlation of SARS-CoV-2 RNA in wastewater with COVID-19 disease burden in sewersheds.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease (COVID-19), is shed in feces and the viral ribonucleic acid (RNA) is detectable in wastewater. A nine-week wastewater epidemiology study of ten wastewater facilities, serving 39% of the state of Utah or 1.26 M individuals was conducted in April and May of 2020. COVID-19 cases were tabulated from within each sewershed boundary. RNA from SARS-CoV-2 was detectable in 61% of 126 wastewater samples. Urban sewersheds serving >100,000 individuals and tourist communities had higher detection frequencies. An outbreak of COVID-19 across two communities positively correlated with an increase in wastewater SARS-CoV-2 RNA, while a decline in COVID-19 cases preceded a decline in RNA. SARS-CoV-2 RNA followed a first order decay rate in wastewater, while 90% of the RNA was present in the liquid phase of the influent. Infiltration and inflow, virus decay and sewershed characteristics should be considered during correlation analysis of SAR-CoV-2 with COVID-19 cases. These results provide evidence of the utility of wastewater epidemiology to assist in public health responses to COVID-19.