ABSTRACT
The sequencing of human virus genomes from wastewater samples is an efficient method for tracking viral transmission and evolution at the community level. However, this requires the recovery of viral nucleic acids of high quality. We developed a reusable tangential-flow filtration system to concentrate and purify viruses from wastewater for whole-genome sequencing. A pilot study was conducted with 94 wastewater samples from four local sewersheds, from which viral nucleic acids were extracted, and the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was sequenced using the ARTIC V4.0 primers. Our method yielded a high probability (0.9) of recovering complete or near-complete SARS-CoV-2 genomes (>90% coverage at 10x depth) from wastewater when the COVID-19 incidence rate exceeded 33 cases per 100 000 people. The relative abundances of sequenced SARS-CoV-2 variants followed the trends observed from patient-derived samples. We also identified SARS-CoV-2 lineages in wastewater that were underrepresented or not present in the clinical whole-genome sequencing data. The developed tangential-flow filtration system can be easily adopted for the sequencing of other viruses in wastewater, particularly those at low concentrations. SYNOPSISThe tangential-flow filtration method extracts viral nucleic acids of high enough quality from wastewater for robust and successful whole-genome sequencing. GRAPHIC FOR TABLE OF CONTENTS (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/22279692v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@566377org.highwire.dtl.DTLVardef@19c3ba7org.highwire.dtl.DTLVardef@106c70org.highwire.dtl.DTLVardef@3f3f8f_HPS_FORMAT_FIGEXP M_FIG C_FIG
ABSTRACT
BackgroundRecent applications of wastewater-based epidemiology (WBE) have demonstrated its ability to track the spread and dynamics of COVID-19 at the community level. Despite the growing body of research, quantitative synthesis of SARS-CoV-2 titers in wastewater generated from studies across space and time using diverse methods has not been performed. ObjectiveThe objective of this study is to examine the correlations between SARS-CoV-2 viral titers in wastewater across studies, stratified by key covariates in study methodologies. In addition, we examined the associations of proportions of positive detections (PPD) in wastewater samples and methodological covariates. MethodsWe systematically searched the Web of Science for studies published by February 16th, 2021, performed a reproducible screen, and employed mixed-effects models to estimate the levels of SARS-CoV-2 viral titers in wastewater samples and their correlations to case prevalence, sampling mode (grab or composite sampling), and the fraction of analysis (FOA, i.e., solids, solid-supernatant mixtures, or supernatants/filtrates) ResultsA hundred and one studies were found; twenty studies (1,877 observations) were retained following a reproducible screen. The mean of PPD across all studies was 0.67 (95%-CI, [0.56, 0.79]). The mean titer was 5,244.37 copies/mL (95%-CI, [0; 16,432.65]). The Pearson Correlation coefficients (PCC) between viral titers and case prevalences were 0.28 (95%-CI, [0.01; 0.51) for daily new cases or 0.29 (95%-CI, [-0.15; 0.73]) for cumulative cases. FOA accounted for 12.4% of the variability in PPD, followed by case prevalence (9.3% by daily new cases and 5.9% by cumulative cases) and sampling mode (0.6%). Among observations with positive detections, FOA accounted for 56.0% of the variability in titers, followed by sampling mode (6.9%) and case prevalence (0.9% by daily new cases and 0.8% by cumulative cases). While sampling mode and FOA both significantly correlated with SARS-CoV-2 titers, the magnitudes of increase in PPD associated with FOA were larger. Mixed-effects model treating studies as random effects and case prevalence as fixed effects accounted for over 90% of the variability in SARS-CoV-2 PPD and titers. InterpretationsPositive pooled means and confidence intervals in PCC between SARS-CoV-2 titers and case prevalence indicators provide quantitative evidence reinforcing the value of wastewater-based monitoring of COVID-19. Large heterogeneities among studies in proportions of positive detections, titers, and PCC suggest a strong demand in methods to generate data accounting for cross-study heterogeneities and more detailed metadata reporting. Large variance explained by FOA suggesting FOA as a direction that needs to be prioritized in method standardization. Mixed-effects models accounting for study level variations provide a new perspective to synthesize data from multiple studies.
