Impact of coagulation on SARS-CoV-2 and PMMoV viral signal in wastewater solids (preprint)

Wastewater surveillance (WWS) has received interest from researchers, scientists, and public health units for its application in monitoring active COVID-19 cases and detecting outbreaks. While WWS of SARS-CoV-2 has been widely applied worldwide, a knowledge gap exists concerning the effects of enhanced primary clarification, the application of coagulant to primary clarifiers, on SARS-CoV-2 and PMMoV quantification for reliable wastewater-based epidemiology. Ferric-based chemical coagulants are extensively used in enhanced clarification, particularly for phosphorus removal, in North America, and Europe. This study examines the effects of coagulation with ferric sulfate on the measurement of SARS-CoV-2 and PMMoV viral measurements in wastewater primary sludge and hence also settled solids. The addition of Fe3+ to wastewater solids ranging from 0 to 60 mg/L caused no change in N1 and N2 gene region measurements in wastewater solids, where Fe3+ concentrations in primary clarified sludge represent the conventional minimum and maximum concentrations of applied ferric-based coagulant. However, elevated Fe3+ concentrations were shown to be associated with a statistically significant increase in PMMoV viral measurements in wastewater solids, which consequently resulted in the underestimation of PMMoV normalized SARS-CoV-2 viral signal measurements (N1 and N2 copies/copies of PMMoV). pH reduction from coagulant addition did not contribute to the increase in PMMoV measurements. Thus, this phenomenon is likely attributed to the partitioning of PMMoV particles to the solids of wastewater from the bulk liquid phase of wastewater.

Tracing COVID-19 Trails in Wastewater: A Systematic Review of SARS-CoV-2 Surveillance with Viral Variants

The emergence of new variants of SARS-CoV-2 associated with varying infectivity, pathogenicity, diagnosis, and effectiveness against treatments challenged the overall management of the COVID-19 pandemic. Wastewater surveillance (WWS), i.e., monitoring COVID-19 infections in communities through detecting viruses in wastewater, was applied to track the emergence and spread of SARS-CoV-2 variants globally. However, there is a lack of comprehensive understanding of the use and effectiveness of WWS for new SARS-CoV-2 variants. Here we systematically reviewed published articles reporting monitoring of different SARS-CoV-2 variants in wastewater by following the PRISMA guidelines and provided the current state of the art of this study area. A total of 80 WWS studies were found that reported different monitoring variants of SARS-CoV-2 until November 2022. Most of these studies (66 out of the total 80, 82.5%) were conducted in Europe and North America, i.e., resource-rich countries. There was a high variation in WWS sampling strategy around the world, with composite sampling (50/66 total studies, 76%) as the primary method in resource-rich countries. In contrast, grab sampling was more common (8/14 total studies, 57%) in resource-limited countries. Among detection methods, the reverse transcriptase polymerase chain reaction (RT-PCR)-based sequencing method and quantitative RT-PCR method were commonly used for monitoring SARS-CoV-2 variants in wastewater. Among different variants, the B1.1.7 (Alpha) variant that appeared earlier in the pandemic was the most reported (48/80 total studies), followed by B.1.617.2 (Delta), B.1.351 (Beta), P.1 (Gamma), and others in wastewater. All variants reported in WWS studies followed the same pattern as the clinical reporting within the same timeline, demonstrating that WWS tracked all variants in a timely way when the variants emerged. Thus, wastewater monitoring may be utilized to identify the presence or absence of SARS-CoV-2 and follow the development and transmission of existing and emerging variants. Routine wastewater monitoring is a powerful infectious disease surveillance tool when implemented globally.

Wastewater surveillance of influenza activity: Early detection, surveillance, and subtyping in city and neighbourhood communities (preprint)

Recurrent epidemics of influenza infection and its pandemic potential present a significant risk to global population health. To mitigate hospitalizations and death, local public health relies on clinical surveillance to locate and monitor influenza-like illnesses and/or influenza cases and outbreaks. At an international level, the global integration of clinical surveillance networks is the only reliable method to report influenza types and subtypes and warn of an emergent pandemic strain. During the COVID-19 pandemic, the demonstrated utility of wastewater surveillance (WWS) in complementing or even replacing clinical surveillance, the latter a resource-intensive enterprise, was predicated on the presence of stable viral fragments in wastewater. We show that influenza virus targets are stable in wastewaters and partitions to the solids fraction. We subsequently quantify, type, and subtype influenza virus in municipal wastewater and primary sludge throughout the course of a community outbreak. This research demonstrates the feasibility of applying influenza virus WWS to city and neighbourhood levels; showing a 17-day lead time in forecasting a citywide flu outbreak and providing population-level viral subtyping in near real-time using minimal resources and infrastructure.

First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan (preprint)

Wastewater-based epidemiology is a powerful tool to understand the actual incidence of coronavirus disease 2019 (COVID-19) in a community because severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can be shed in the feces of infected individuals regardless of their symptoms. The present study aimed to assess the presence of SARS-CoV-2 RNA in wastewater and river water in Yamanashi Prefecture, Japan, using four quantitative and two nested PCR assays. Influent and secondary-treated (before chlorination) wastewater samples and river water samples were collected five times from a wastewater treatment plant and three times from a river, respectively, between March 17 and May 7, 2020. The wastewater and river water samples (200-5,000 mL) were processed by using two different methods: the electronegative membrane-vortex (EMV) method and the membrane adsorption-direct RNA extraction method. Based on the observed concentrations of indigenous pepper mild mottle virus RNA, the EMV method was found superior to the membrane adsorption-direct RNA extraction method. SARS-CoV-2 RNA was successfully detected in one of five secondary-treated wastewater samples with a concentration of 2.4 x 103 copies/L by N_Sarbeco qPCR assay following the EMV method, whereas all the influent samples were tested negative for SARS-CoV-2 RNA. This result could be attributed to higher limit of detection for influent (4.0 x 103-8.2 x 104 copies/L) with a lower filtration volume (200 mL) compared to that for secondary-treated wastewater (1.4 x 102-2.5 x 103 copies/L) with a higher filtration volume of 5,000 mL. None of the river water samples tested positive for SARS-CoV-2 RNA. Comparison with the reported COVID-19 cases in Yamanashi Prefecture showed that SARS-CoV-2 RNA was detected in the secondary-treated wastewater sample when the cases peaked in the community. This is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in Japan.