Biomarkers Selection for Population Normalization in SARS-CoV-2 Wastewater-based Epidemiology

Abstract

Wastewater-based epidemiology (WBE) has been one of the most cost-effective approaches to track the SARS-CoV-2 levels in the communities since the COVID-19 outbreak in 2020. Normalizing SARS-CoV-2 concentrations by the population biomarkers in wastewater can be critical for interpreting the viral loads, comparing the epidemiological trends among the sewersheds, and identifying the vulnerable communities. In this study, five population biomarkers, pepper mild mottle virus (pMMoV), creatinine (CRE), 5-hydroxyindoleacetic acid (5-HIAA), caffeine (CAF) and its metabolite paraxanthine (PARA) were investigated for their utility in normalizing the SARS-CoV-2 loads through developed direct and indirect approaches. Their utility in assessing the real-time population contributing to the wastewater was also evaluated. The best performed candidate was further tested for its capacity for improving correlation between normalized SARS-CoV-2 loads and the clinical cases reported in the City of Columbia, Missouri, a university town with a constantly fluctuated population. Our results showed that, except CRE, the direct and indirect normalization approaches using biomarkers allow accounting for the changes in wastewater dilution and differences in relative human waste input over time regardless flow volume and population at any given WWTP. Among selected biomarkers, PARA is the most reliable population biomarker in determining the SARS-CoV-2 load per capita due to its high accuracy, low variability, and high temporal consistency to reflect the change in population dynamics and dilution in wastewater. It also demonstrated its excellent utility for real-time assessment of the population contributing to the wastewater. In addition, the viral loads normalized by the PARA-estimated population significantly improved the correlation (rho=0.5878, p<0.05) between SARS-CoV-2 load per capita and case numbers per capita. This chemical biomarker offers an excellent alternative to the currently CDC-recommended pMMoV genetic biomarker to help us understand the size, distribution, and dynamics of local populations for forecasting the prevalence of SARS-CoV2 within each sewershed.

Population Normalization in SARS-CoV-2 Wastewater-Based Epidemiology: Implications from Statewide Wastewater Monitoring in Missouri

“…In addition, our previous study confirmed that direct normalization effects of SARS-CoV-2 concentration using biomarker concentrations were always less ideal than indirect normalization, which involved the wastewater volume. 29…”

“…In addition, our previous study confirmed that direct normalization effects of SARS-CoV-2 concentration using biomarker concentrations were always less ideal than indirect normalization, which involved the wastewater volume. 29 Our findings suggest that for long-term wastewater SARS-CoV-2 surveillance, normalizing SARS-CoV-2 wastewater concentrations with a reliable population marker prior to calculating trends is highly recommended to account for changes in wastewater dilution and differences in relative human waste input over time due to tourism, weekday commuters, temporary workers, and pandemic lockdowns, etc. This approach is particularly critical for the sewershed with dynamic population changes, such as colleges, tourist towns, and metropolitan areas, where a large number of commuters who used to travel to cities daily transitioned to fully or partially remote workers after the pandemic.…”

“…The extracted RNA was used to perform RT-qPCR quantification of the genetic material of SARS-CoV-2 and PMMoV, separately. SARS-CoV-2 was quantified using the primer and control sets described in Robinson et al, 32 and PMMoV was quantified using the primer sets described in Hsu et al 29 A plasmid carrying a PMMoV gene along with an N gene fragment was constructed, purified from Escherichia coli , and used as standards for the RT-qPCR assay. Final RT-qPCR one-step mixtures consisted of 5 µL TaqPath 1-step RT-qPCR Master Mix (Thermo Fisher), 500 nM of each primer, 125 nM of each TaqMan probes, 5 µl of wastewater RNA extract, and RNase/DNase-free water to reach a final volume of 20 µL.…”

“…28 Our previous study showed that the recovery rates of caffeine and paraxanthine during injection are 101±7% and 92±3%. 29 In addition, the repeatability for caffeine and paraxanthine was quite high (CV%: 12% and 5%). 28 Unlike the simple chemical analysis on LC-MSMS, PMMoV measurement has a very complex workflow.…”

“…In a previous study, we compared five biomarkers, PMMoV, creatinine, 5-HIAA, caffeine, and paraxanthine, based on two weeks’ data, for their utility in normalizing SARS-CoV-2 loads and found caffeine and especially paraxanthine were the most reliable population biomarkers. 29 This study extended the investigation to the long-term weekly monitoring of 64 wastewater treatment plants (WWTPs) across Missouri for more than six months to compare the utility of caffeine, paraxanthine, and PMMoV as population biomarkers across seasons and distinct geographical areas with contrasting sewershed sizes.…”