Development of a reproducible method for monitoring SARS-CoV-2 in wastewater

Abstract

Monitoring the genetic signal of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through RNA titers in wastewater has emerged as a promising strategy for tracking community-scale prevalence of coronavirus disease 2019 (COVID-19). Although many studies of SARS-CoV-2 in wastewater have been conducted around the world, a uniform procedure for concentrating the virus in wastewater is lacking. The goal of this study was to comprehensively evaluate how different methods for concentrating the suspended solids in wastewater affect the associated SARS-CoV-2 RNA signal and the time required for processing samples for wastewater-based epidemiology efforts. We additionally consider the effects of sampling location in the wastewater treatment train (i.e., following preliminary or primary treatment), pasteurization, and RNA extraction method. Comparison of the liquid phase to suspended solids obtained via centrifugation or vacuum filtration suggests that the RNA signal of SARS-CoV-2 preferentially occurs in the solids. Therefore, we assert that the recovery of SARS-CoV-2 from wastewater should focus on suspended solids. Our data indicate that the measured SARS-CoV-2 signal is higher among samples taken from the primary clarifier effluent, as opposed to those taken after preliminary treatment. Additionally, we provide evidence that sample pasteurization at 60 °C for 90 min reduces the SARS-CoV-2 signal by approximately 50-55%. Finally, the results indicate that a magnetic bead approach to RNA extraction leads to a higher SARS-CoV-2 signal than does a silica membrane approach.

Space-time analysis of COVID-19 cases and SARS-CoV-2 wastewater loading: A geodemographic perspective

“… Farkas et al (2020) detailed that viral concentrations in wastewater can provide an essential indicator of viral disease prevalence in a community. While the methods for concentrating and detecting SARS-CoV-2 in wastewater continue to be developed, many researchers have successfully extracted SARS-CoV-2 RNA from untreated sludge samples or other bulk wastewater sampling points ( Balboa et al, 2021 ; Palmer et al, 2021 ). Quantifying the viral concentration in wastewater samples typically involves reverse-transcription, quantitative, real-time polymerase chain reaction (RT-qPCR), digital PCR, or digital-drop PCR (ddPCR) methods.…”

A safe haven of SARS-CoV-2 in the environment: Prevalence and potential transmission risks in the effluent, sludge, and biosolids

“…The SARS-CoV-2 virus and other emerging pollutants are of particular concern since some of the treatment plants were not designed to typically monitor or sufficiently remove them during the wastewater treatment processes, thereby posing great risks to human and environmental health. The basic conventional primary treatment processes, including screens, grit chamber, and primary clarifier that are often deployed in rural settings of developing countries have been reported to lack the capacity to remove or inactivate coronaviruses ( Balboa et al, 2021 ; D’Aoust et al, 2021; Palmer et al, 2021 ).

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“…The coronavirus disease 2019 (COVID-19) pandemic caused by a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) has led to a significant paradigm shift on the existing precaution towards handling and exposure to different environmental materials such as water, air, and soil ( Cao et al, 2021 , Shao et al, 2021 , Anand et al, 2022 ). The prevalence of the SARS-CoV-2 or its genetic material in the environmental materials, especially wastewater samples collected from treatment plants and air samples from the built environment have been well documented in many countries, including the USA ( Nemudryi et al, 2020 , Peccia et al, 2020 , Sherchan et al, 2020 , Palmer et al, 2021 ), Spain ( Randazzo et al, 2020 , Balboa et al, 2021 ), Turkey ( Kocamemi et al, 2020a , Kocamemi et al, 2020b ), Netherlands ( Medema et al, 2020 ), Saudi Arabia ( Hong et al, 2021 ), Australia ( Ahmed et al, 2020 ), Japan ( Haramoto et al, 2020 , Hata et al, 2021 ), Iran ( Gholipour et al, 2021 , Tanhaei et al, 2021 ), Czech Republic ( Mlejnkova et al, 2020 ), India ( Chakraborty et al, 2021 , Kumar et al, 2021a ), France ( Trottier et al, 2020 , Wurtzer et al, 2021 ), United Arab Emirate (Hasan et al, 2020), Germany ( Westhaus et al, 2021 ), Israel ( Abu Ali et al, 2021 ), and Italy (La Rosa et al, 2020; Rimoldi et al, 2020 ). Similarly, some treated effluents have also been reported to contain the SARS-CoV-2 RNA ( Ampuero et al, 2020 , Abu Ali et al, 2021 , Bhattarai et al, 2021 , Carrillo-Reyes et al, 2021 , Graham et al, 2021 ).…”

Monitoring COVID‐19 through SARS‐CoV‐2 quantification in wastewater: progress, challenges and prospects

“…However, other studies have included other methods like the Moore swabs, a gauze pad that is suspended in flowing WW and then processed (Liu et al ., 2020; Rafiee et al ., 2021). Each method may provide different information that will depend on the volume of sample gathered (Kitajima et al ., 2020), the time of the day (Kopperi et al ., 2021) if the sample is not an equalized composite, as well as the point of sampling within the WW treatment train (Balboa et al ., 2021; Palmer et al ., 2021). Grabs can potentially have lower costs and be easier to perform than composite samples but could also have a higher degree of variability.…”

“…Finally, several labs are also including a heat pre‐treatment to reduce SARS‐CoV‐2 virulence in the samples without compromising its quantification, in order to avoid the need for hard safety measures to prevent lab workers infections (Batéjat et al ., 2020; La Rosa et al ., 2020; Pastorino et al ., 2020; Palmer et al ., 2021).…”