Comparing solid-based concentration methods for rapid and efficient recovery of SARS-CoV-2 for wastewater surveillance

As wastewater-based surveillance of SARS-CoV-2 attracts interest globally, there is a need to evaluate and identify rapid and efficient methods for concentrating enveloped viruses in wastewater. When comparing five precipitation/flocculation-based concentration methods (including aluminum hydroxide adsorptionprecipitation, AHAP; zinc acetate precipitation, ZAP; skimmed milk flocculation, SMF; FeCl3 precipitation, FCP; and direct centrifugation, DC), AHAP was found to be the most efficient method in terms of seeded BCoV recovery (50.2 %). Based on the BCoV recovery efficiency and turnaround time, the AHAP and DC methods were selected and tested on five additional wastewater samples containing both seeded BCoV and indigenous wastewater SARS-CoV-2 RNA. The BCoV recovery (DC: average = 30.1 %, sx = 14.7 %; AHAP: average = 33.0 %, sx = 14.2 %) and SARS-CoV-2 based on the N2 gene assay (DC: average = 3.6 x 103 gene copies or GC/mL, sx = 1.9 x 103 GC/mL; AHAP: average=3.0 x 103 GC/mL, sx = 2.0 x 103 GC/mL) of both methods were not significantly different in solid fraction (p = 0.89). This study showed significant higher BCoV recovery and SARSCoV-2 viral RNA in wastewater solid fraction (p = 0.006) than liquid fraction. Our result suggests that the solid fraction of wastewater samples is more suitable for recovering enveloped viruses from wastewater, and the DC and AHAP methods equally provide suitably rapid, cost-effective, and significantly higher recovery of SARS-CoV2 viral RNA in wastewater samples.