Evaluating recovery, cost, and throughput of different concentration methods for SARS-CoV-2 wastewater-based epidemiology

Abstract

As the COVID-19 pandemic continues to affect communities across the globe, the need to contain the spread of the outbreaks is of paramount importance. Wastewater monitoring of the SARS-CoV-2 virus, the causative agent responsible for COVID-19, has emerged as a promising tool for health officials to anticipate outbreaks. As interest in wastewater monitoring continues to grow and municipalities begin to implement this approach, there is a need to further identify and evaluate methods used to concentrate SARS-CoV-2 virus RNA from wastewater samples. Here we evaluate the recovery, cost, and throughput of five different concentration methods for quantifying SARS-CoV-2 virus RNA in wastewater samples. We tested the five methods on six different wastewater samples. We also evaluated the use of a bovine coronavirus vaccine as a process control and pepper mild mottle virus as a normalization factor. Of the five methods we tested head-to-head, we found that HA filtration with bead beating performed the best in terms of sensitivity and cost. This evaluation can serve as a guide for laboratories establishing a protocol to perform wastewater monitoring of SARS-CoV-2.

Evaluation of sampling frequency and normalization of SARS-CoV-2 wastewater concentrations for capturing COVID-19 burdens in the community

“…Our results indicate the HA filter method [30,31] combined with one-step RT-ddPCR is effective for tracking SARS-CoV-2 concentrations in wastewater influent. Similarly to reports by Altruners [17], we found the HA filter method was relatively fast and allowed an effective analysis volume for each ddPCR reaction (∼1.5 mL), which provided the needed sensitivity to quantify SARS-CoV-2 when reported cases were as low as 9 per 100,000 people in the watershed. We also found that the N1 assay was more sensitive than the N2 assay when levels were near the limits of detection, likely because of assay efficiency.…”

“…One major challenge to implementing widespread SARS-CoV-2 wastewater surveillance is the lack of a standard method. Enveloped viruses partition between the solid and liquid phases in waste streams [16] and research is underway to evaluate concentration methods for these viruses in wastewater [17,18]. Using common controls can allow for comparison of results across different methods or laboratories [19].…”

“…Using common controls can allow for comparison of results across different methods or laboratories [19]. A variety of surrogates have been used to estimate recovery, including bovine coronavirus (BCoV) [12,17], bovine respiratory syncytial virus (BRSV) [20], and human coronavirus (OC43) [21]. In addition, normalizing SARS-CoV-2 RNA concentrations to WWTP flow, the estimated amount of human fecal material in the samples (the fecal strength), and/or the population contributing to the viral load is needed to compare across samples and WWTPs.…”

Within-Day Variability of SARS-CoV-2 RNA in Municipal Wastewater Influent During Periods of Varying COVID-19 Prevalence and Positivity

“…This is consistent with the improved recoveries observed for BRSV after solids were removed. Despite the low and variable recovery in the current study, adsorption-extraction using electronegative membranes demonstrated reproducibility and sensitivity at reasonable cost in two previous methods comparisons 42,51 .…”

Sewage, Salt, Silica, and SARS-CoV-2 (4S): An Economical Kit-Free Method for Direct Capture of SARS-CoV-2 RNA from Wastewater

“… 15 These methods can be relatively time-consuming and become inaccessible if they are dependent on reliable supply of commercial reagents, a paucity of which has already hampered clinical SARS-CoV-2 testing efforts. 13 − 17 Furthermore, the use of primary concentration assumes the recovery of intact virus and is therefore not geared toward cocapturing RNA from SARS-CoV-2 viruses that have already lysed or capture of nonviral RNAs suitable as fecal concentration controls. Lastly, current CDC safety guidelines recommend a biosafety level 2 facility with unidirectional airflow and BSL-3 precautions when employing environmental sampling procedures that concentrate viruses presumed to be intact.…”