Persistence of human respiratory viral RNA in wastewater-settled solids.

Wastewater-based epidemiology has emerged as a valuable tool for monitoring respiratory viral diseases within communities by analyzing concentrations of viral nucleic-acids in wastewater. However, little is known about the fate of respiratory virus nucleic-acids in wastewater. Two important fate processes that may modulate their concentrations in wastewater as they move from household drains to the point of collection include sorption or partitioning to wastewater solids and degradation. This study investigated the decay kinetics of genomic nucleic-acids of seven human respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV), human coronavirus (HCoV)-OC43, HCoV-229E, HCoV-NL63, human rhinovirus (HRV), and influenza A virus (IAV), as well as pepper mild mottle virus (PMMoV) in wastewater solids. Viruses (except for PMMoV) were spiked into wastewater solids and their concentrations were followed for 50 days at three different temperatures (4°C, 22°C, and 37°C). Viral genomic RNA decayed following first-order kinetics with decay rate constants k from 0 to 0.219 per day. Decay rate constants k were not different from 0 for all targets in solids incubated at 4°C; k values were largest at 37°C and at this temperature, k values were similar across nucleic-acid targets. Regardless of temperature, there was limited viral RNA decay, with an estimated 0% to 20% reduction, over the typical residence times of sewage in the piped systems between input and collection point (<1 day). The k values reported herein can be used directly in fate and transport models to inform the interpretation of measurements made during wastewater surveillance.IMPORTANCEUnderstanding whether or not the RNA targets quantified for wastewater-based epidemiology (WBE) efforts decay during transport between drains and the point of sample collection is critical for data interpretation. Here we show limited decay of viral RNA targets typically measured for respiratory disease WBE.

Adsorption of Respiratory Syncytial Virus, Rhinovirus, SARS-CoV-2, and F+ Bacteriophage MS2 RNA onto Wastewater Solids from Raw Wastewater.

Despite the widespread adoption of wastewater surveillance, more research is needed to understand the fate and transport of viral genetic markers in wastewater. This information is essential for optimizing monitoring strategies and interpreting wastewater surveillance data. In this study, we examined the solid-liquid partitioning behavior of four viruses in wastewater: SARS-CoV-2, respiratory syncytial virus (RSV), rhinovirus (RV), and F+ coliphage/MS2. We used two approaches: (1) laboratory partitioning experiments using lab-grown viruses and (2) distribution experiments using endogenous viruses in raw wastewater. Partition experiments were conducted at 4 and 22 °C. Wastewater samples were spiked with varying concentrations of each virus, solids and liquids were separated via centrifugation, and viral RNA concentrations were quantified using reverse-transcription-digital droplet PCR (RT-ddPCR). For the distribution experiments, wastewater samples were collected from six wastewater treatment plants and processed without spiking exogenous viruses; viral RNA concentrations were measured in wastewater solids and liquids. In both experiments, RNA concentrations were higher in the solid fraction than the liquid fraction by approximately 3-4 orders of magnitude. Partition coefficients (KF) ranged from 2000-270,000 mL·g-1 across viruses and temperature conditions. Distribution coefficients (Kd) were consistent with results from partitioning experiments. Further research is needed to understand how virus and wastewater characteristics might influence the partitioning of viral genetic markers in wastewater.

Persistence of Endogenous SARS-CoV-2 and Pepper Mild Mottle Virus RNA in Wastewater-Settled Solids.

Limited information is available on the decay rate of endogenous SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA in wastewater and primary settled solids, potentially limiting an understanding of how transit or holding times within wastewater infrastructure might impact RNA measurements and their relationship to community COVID-19 infections. In this study, primary settled solids samples were collected from two wastewater treatment plants in the San Francisco Bay Area. Samples were thoroughly mixed, aliquoted into subsamples, and stored at 4, 22, and 37 °C for 10 days. The concentrations of SARS-CoV-2 (N1 and N2 targets) and PMMoV RNA were measured using an RT-ddPCR. Limited decay (<1 log10 reduction) was observed in the detection of viral RNA targets at all temperature conditions, suggesting that SARS-CoV-2 and PMMoV RNA can be highly persistent in solids. First-order decay rate constants ranged from 0.011 to 0.098 day-1 for SARS-CoV-2 RNA and from 0.010 to 0.091 day-1 for PMMoV RNA depending on the temperature conditions. A slower decay was observed for SARS-CoV-2 RNA in primary settled solids compared to previously reported decay in wastewater influent. Further research is needed to understand if solid content and wastewater characteristics might influence the persistence of viral RNA targets.

Scaling of SARS-CoV-2 RNA in Settled Solids from Multiple Wastewater Treatment Plants to Compare Incidence Rates of Laboratory-Confirmed COVID-19 in Their Sewersheds.

Published and unpublished reports show that SARS-CoV-2 RNA in publicly owned treatment work (POTW) wastewater influent and solids is associated with new COVID-19 cases or incidence in associated sewersheds, but methods for comparing data collected from diverse POTWs to infer information about the relative incidence of laboratory-confirmed COVID-19 cases, and scaling to allow such comparisons, have not been previously established. Here, we show that SARS-CoV-2 N1 and N2 concentrations in solids normalized by concentrations of PMMoV RNA in solids can be used to compare incidence of laboratory confirmed new COVID-19 cases across POTWs. Using data collected at seven POTWs along the United States West Coast, Midwest, and East Coast serving ∼3% of the U.S. population (9 million people), we show that a 1 log change in N gene/PMMoV is associated with a 0.24 (range 0.19 to 0.29) log10 change in incidence of laboratory confirmed COVID-19. Scaling of N1 and N2 by PMMoV is consistent, conceptually, with a mass balance model relating SARS-CoV-2 RNA to the number of infected individuals shedding virus in their stool. This information should support the application of wastewater-based epidemiology to inform the response to the COVID-19 pandemic and potentially future viral pandemics.

SARS-CoV-2 RNA in Wastewater Settled Solids Is Associated with COVID-19 Cases in a Large Urban Sewershed.

Wastewater-based epidemiology may be useful for informing public health response to viral diseases like COVID-19 caused by SARS-CoV-2. We quantified SARS-CoV-2 RNA in wastewater influent and primary settled solids in two wastewater treatment plants to inform the preanalytical and analytical approaches and to assess whether influent or solids harbored more viral targets. The primary settled solids samples resulted in higher SARS-CoV-2 detection frequencies than the corresponding influent samples. Likewise, SARS-CoV-2 RNA was more readily detected in solids using one-step digital droplet (dd)RT-PCR than with two-step RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal time series of 89 settled solids samples from a single plant for SARS-CoV-2 RNA as well as coronavirus recovery (bovine coronavirus) and fecal strength (pepper mild mottle virus) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlated positively and significantly with COVID-19 clinically confirmed case counts in the sewershed. Together, the results demonstrate that measuring SARS-CoV-2 RNA concentrations in settled solids may be a more sensitive approach than measuring SARS-CoV-2 in influent.