Comparison of adsorption-extraction workflows for improved measurements of viruses and bacteria in untreated wastewater.

The lack of standardized methods and large differences in virus concentration and extraction workflows have hampered severe acute respiratory syndrome (SARS-CoV-2) wastewater surveillance and data reporting practices. Numerous studies have shown that adsorption-extraction (AE) method holds promise, yet several uncertainties remain regarding the optimal AE workflow. Several procedural components that may influence the recovered concentrations of target DNA/RNA, including membrane types, homogenization instruments, speed and duration, and lysis buffer. In this study, 42 different AE workflows that varied these components were compared to determine the optimal method by quantifying endogenous SARS-CoV-2, human adenovirus (HAdV 40/41) and a bacterial marker gene of fecal pollution (Bacteroides HF183). Our findings suggest that the certain selected workflow had a significant impact on SARS-CoV-2 concentrations, whereas it had minimal impact on HF183 and no effect on HAdV 40/41 concentrations. When comparing individual components in a workflow, such as membrane type (MF-Millipore™ 0.45 μm MCE vs. Isopore™ 0.40 μm) and homogenization instruments (Precellys 24 homogenizer vs. Vortex-Genie®-2), we found that they had no impact on SARS-CoV-2, HAdV 40/41, and HF183 concentrations. This suggests that at least some consumables and equipment are interchangeable. Buffer PM1 + TRIzol based workflows yielded higher concentrations of SARS-CoV-2 than other workflows. HF183 concentrations were higher in workflows without chloroform. Similarly, higher homogenization speeds (5000-10,000 rpm) led to increased concentrations of SARS-CoV-2 and HF183 but had no effect on HAdV 40/41. Our findings indicate that minor enhancements to the AE workflow can improve the recovery of viruses and bacteria from the wastewater, leading to different outcomes from wastewater surveillance efforts.