Co-quantification of crAssphage increases confidence in wastewater-based epidemiology for SARS-CoV-2 in low prevalence areas

Abstract

Wastewater surveillance of SARS-CoV-2 RNA is increasingly being incorporated into public health efforts to respond to the COVID-19 pandemic. In order to obtain the maximum benefit from these efforts, approaches to wastewater monitoring need to be rapid, sensitive, and relatable to relevant epidemiological parameters. In this study, we present an ultracentrifugation-based method for the concentration of SARS-CoV-2 wastewater RNA and use crAssphage, a bacteriophage specific to the human gut, to help account for RNA loss during transit in the wastewater system and sample processing. With these methods, we were able to detect, and sometimes quantify, SARS-CoV-2 RNA from 20 mL wastewater samples within as little as 4.5 hours. Using known concentrations of bovine coronavirus RNA and deactivated SARS-CoV-2, we estimate recovery rates of approximately 7-12% of viral RNA using our method. Results from 24 sewersheds across Upstate New York during the spring and summer of 2020 suggested that stronger signals of SARS-CoV-2 RNA from wastewater may be indicative of greater COVID-19 incidence in the represented service area approximately one week in advance. SARS-CoV-2 wastewater RNA was quantifiable in some service areas with daily positives tests of less than 1 per 10,000 people or when weekly positive test rates within a sewershed were as low as 1.7%. crAssphage DNA concentrations were significantly lower during periods of high flow in almost all areas studied. After accounting for flow rate and population served, crAssphage levels per capita were estimated to be about 1.35 × 10 11 and 2.42 × 10 8 genome copies per day for DNA and RNA, respectively. A negative relationship between per capita crAssphage RNA and service area size was also observed likely reflecting degradation of RNA over long transit times. Our results reinforce the potential for wastewater surveillance to be used as a tool to supplement understanding of infectious disease transmission obtained by traditional testing and highlight the potential for crAssphage co-detection to improve interpretations of wastewater surveillance data.

Tools for interpretation of wastewater SARS-CoV-2 temporal and spatial trends demonstrated with data collected in the San Francisco Bay Area

“…Pepper mild mottle virus (PMMoV), a nonenveloped RNA plant virus, is commonly used for COVID-19 WBE ( D'Aoust et al, 2021b ; Feng et al, 2021 ; Whitney et al, 2021 ; Wu et al, 2020 ) but concentrations in sewage vary with season and local diet ( Symonds et al, 2019 ). Another normalization biomarker is the cross-assembly phage (crAssphage), a non-enveloped, DNA virus that ubiquitously infects the human gut commensal bacteria Bacteroides ( Edwards et al, 2019 ; Green et al, 2020 ; Stachler et al, 2017 ; Wilder et al, 2021 ). In addition, Bacteroides HF183 16S rRNA gene is widely used for detecting fecal contamination in environmental waters ( Green et al, 2020 ; Shanks et al, 2008 ), and recent studies ( D'Aoust et al, 2021b ; Kapoor et al, 2015 ; Pitkänen et al, 2013 ) have targeted HF183 rRNA (versus the rRNA gene) to increase the sensitivity of the assay ( D'Aoust et al, 2021b ; Feng et al, 2021 ).…”

Technical framework for wastewater-based epidemiology of SARS-CoV-2

“…In addition, reasonably selected reference gene itself is a good internal control of the whole method, which can exclude the influence of concentration efficiency, inhibiting substance of PCR and other factors to a certain extent ( Jafferali et al, 2021 ). A recent study has shown that not only the DNA of crAssphage, but also its transcription product RNA can be detected in sewage ( Wilder et al, 2021 ). Therefore, crAssphage has the potential as a dual reference biomarker for simultaneously detecting RNA and DNA viruses.…”

Efficacy of SARS-CoV-2 wastewater surveillance for detection of COVID-19 at a residential private college

“…RNA was extracted following the method of Wilder et al 2021 (22). All steps prior to RNA extraction were conducted in BSL2 conditions, as approved by the Colgate University Institutional Biosafety Committee.…”