Wastewater-based protocols for SARS-CoV-2: insights into virus concentration, extraction, and quantitation methods from two years of public health surveillance†

The ongoing COVID-19 pandemic has accelerated the development and application of wastewater-based disease surveillance (WBS) as a tool for public health practice. The wide variety of WBS methods currently in use hinders the ability to compare data between different laboratories and limits the potential of nationwide surveillance programs. In this study, we conducted a systematic analysis to identify among widely used concentration, extraction and quantification methods, which ones would perform well for WBS of SARS-CoV-2. We evaluated electronegative filtration, one of the traditional methods applied early in the pandemic, to other methods including direct capture, magnetic affinity particles and PEG. Our results indicated that these alternative concentration methods quantify SARS-CoV-2 just as effective if not better compared to membrane filtration. We also identified the effect that filtration flow rate, volume filtered, and bead beating parameters have on viral target recovery. The evaluation of different extraction methods demonstrated that an automatic paramagnetic bead-based method performs better than the column-based method tested. In addition, we compared the quantification between RT-qPCR and RT-dPCR, and while both perform well, we documented that RT-dPCR has a lower LOD and can provide more accurate data. Lastly, we compared three weeks of side-by-side wastewater surveillance by two different, but currently commonly applied approaches: HA filtration quantified by RT-qPCR and Ceres Nanotrap® Microbiome A Particles quantified by RT-dPCR. On average, we found a 3.6-fold difference in SARS-CoV-2 levels between the two approaches and observed that the N1 : N2 ratio was closer to one with Nanotrap® particle concentration quantified by RT-dPCR.