Sources of variability in methods for processing, storing, and concentrating SARS-CoV-2 in influent from urban wastewater treatment plants

The rapid emergence of wastewater based surveillance has led to a wide array of SARS-CoV-2 RNA quantification methodologies being employed. Here we compare methods to store samples, inactivate viruses, capture/concentrate viruses, and extract/measure viral RNA from primary influent into wastewater facilities. We found that heat inactivation of the viruses led to a 1-3 log10 decrease compared to chemical inactivation. Freezing influent prior to concentration caused a 1-4 log10 decrease compared to processing fresh samples, but viral capture by membrane adsorption prior to freezing was robust to freeze-thaw variability. Concentration vs. direct extraction, and PCR platform also affected outcome, but by a smaller amount. The choice of nucleocapsid gene target had nearly no effect. Pepper mild-mottle virus was much less sensitive to these methodological differences than was SARS-CoV-2, which challenges its use as a population-level control among studies using different methods. Better characterizing the variability associated with different methodologies, in particular the impact of methods on sensitivity, will aid decision makers in following the effects of vaccination campaigns, early detection of future outbreaks, and potentially monitoring the appearance of SARS-CoV-2 variants in the population.