Wastewater surveillance using ddPCR accurately tracked Omicron emergence due to altered N1 probe binding efficiency

The emergence of Omicron in routine wastewater surveillance was accurately reflected using droplet digital (dd)PCR due to a mutation in the N1 probe binding region that caused diminished fluorescence within individual droplets. Wastewater from five cities provided a higher resolution of Omicron emergence when comparing data from sequenced clinical samples.

Effect of Time and Temperature on SARS-CoV-2 in Municipal Wastewater Conveyance Systems

Wastewater surveillance for SARS-CoV-2 is becoming a widespread public health metric, but little is known about pre-analytical influences on these measurements. We examined SARS-CoV-2 loads from two sewer service areas with different travel times that were within the same metropolitan area. Throughout the one-year study, case rates were nearly identical between the two service areas allowing us to compare differences in empirical concentrations relative to conveyance system characteristics and wastewater treatment plant parameters. We found time did not have a significant effect on degradation of SARS-CoV-2 when using average transit times (22 vs. 7.5 h) (p = 0.08), or under low flow conditions when transit times are greater (p = 0.14). Flow increased rather than decreased SARS-CoV-2 case-adjusted concentrations, but this increase was only significant in one service area. Warmer temperatures (16.8–19.8 °C) compared with colder (8.4–12.3 °C) reduced SARS-CoV-2 case-adjusted loads by ~50% in both plants (p < 0.05). Decreased concentrations in warmer temperatures may be an important factor to consider when comparing seasonal dynamics. Oxygen demand and suspended solids had no significant effect on SARS-CoV-2 case-adjusted loads overall. Understanding wastewater conveyance system influences prior to sample collection will improve comparisons of regional or national data for SARS-CoV-2 community infections.

SARS-CoV-2 RNA is enriched by orders of magnitude in primary settled solids relative to liquid wastewater at publicly owned treatment works.

Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Raw wastewater consists of small particles, or solids, suspended in liquid. Methods have been developed to measure SARS-CoV-2 RNA in the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in the solid fraction. To investigate this relationship further, six laboratories collaborated to conduct a study across five publicly owned treatment works (POTWs) where both primary settled solids obtained from primary clarifiers and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Settled solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations, on a mass equivalent basis, were higher in settled solids than in influent by approximately three orders of magnitude. Concentrations in matched settled solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both settled solids and influent were correlated to COVID-19 incidence rates in the sewersheds and thus representative of disease occurrence; the settled solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Settled solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of settled solids for SARS-CoV-2 RNA has the advantage of using less sample volume to achieve similar sensitivity to influent methods.