Impact of wastewater characteristics and weather events on the N2 and N1 gene target ratios during wastewater surveillance of SARS-CoV-2 at five treatment plants and an upper sewershed location

Recent fluctuations in the N2/N1 gene target ratios of SARS-CoV-2 were observed in wastewater, even when the dominant variant remained unchanged. This suggests that N2/N1 variations are influenced not only by viral mutations but also by external factors. While previous studies examined the effects of wastewater parameters on SARS-CoV-2 concentrations, there remains a knowledge gap regarding their specific impact on different gene targets. The main objective of this study was to identify external factors that contribute to the observed changes in the N2/N1 ratios in wastewater apart from reduced PCR sensitivity caused by mutations in the variants of concern. Examined factors included wastewater characteristics (pH, wastewater temperature, total and volatile solids, and turbidity) and weather events (precipitation, snow cover, and ambient temperature). Composite samples were collected over a 17-month period from five wastewater treatment plants and an upper sewershed location, spanning several seasons and weather events. SARS-CoV-2 RNA concentrations were measured using RT-qPCR using the N1 and N2 gene targets, and advanced statistical analyses were applied to assess trends and correlations. The results indicate that wastewater characteristics and weather events significantly impact the N2/N1 ratios, with additional effects from sewer size, type, and location. No single parameter consistently impacted the N2/N1 ratio across all sites, rather, impacts were site-specific. However, volatile solids and turbidity showed consistent correlations with N2/N1 ratios at all wastewater treatment plants. Understanding these external impacts is essential for accurately interpreting the changes in N2/N1 ratios and improving wastewater-based epidemiology efforts.