Comparison of viral concentration techniques for native fecal indicators and pathogens from wastewater

Viral pathogens are typically dilute in environmental waters, necessitating a concentration step prior to subsequent quantification or analysis. Historically, studies on viral concentration efficiency have been done by spiking known viruses into the sample; however, spike-in controls may not have the same behavior as "native" viruses exposed to environmental conditions. In this study, four concentration methods, including polyethylene glycol precipitation (PEG), skimmed milk flocculation (SMF), pH drop followed by filtration through a 0.45 μm filter (pH), and centrifugation using an Amicon filter (Amicon), were evaluated to concentrate native viral targets in wastewater. Viral targets included both indicators (crAssphage and pepper mild mottle virus) and pathogens (adenovirus, norovirus GII, human polyomavirus, and SARS-CoV-2) in addition to a bacterial marker (HF183). A non-native spike-in control was also added to compare native and spike-in recoveries. Recovery varied widely across targets and methods, ranging from 0.1 to 39.3 %. The Amicon method was the most broadly effective concentration for recovery efficiency. For the lowest-titer target, the PEG method resulted in the lowest number of non-detections, with 96.7 % positive detections for SARS-CoV-2, compared to 66.7 %, 80 %, and 76.7 % positive detections for SMF, pH, and Amicon, respectively. The non-native spike-ins chosen were only representative of a few native recovery trends, varying by both target and concentration method, and consistently under or over-estimated recovery. Overall, this study suggests the utility of including native targets in viral concentration evaluation and determining the efficiency of concentration methods for a specific target of interest.