Comparison of RT-qPCR and Digital PCR Methods for Wastewater-Based Testing of SARS-CoV-2 (preprint)

Wastewater-based epidemiology is an important tool for monitoring SARS-CoV-2 and other molecular targets in populations, using wastewater as a pooled sample. We compared the sensitivity, susceptibility to inhibition, and quantification of reverse transcription quantitative PCR (RT-qPCR), microfluidic well digital RT-PCR (RT-dPCR), and droplet digital RT-PCR (RT-ddPCR) measurements of SARS-CoV-2 (N1 gene target) and Pepper Mild Mottle Virus (PMMoV) RNA in 40 wastewater RNA extracts. All three methods were highly sensitive, but appeared less accurate at very low concentrations. Lower inhibition was observed for RT-ddPCR than RT-qPCR with both SARS-CoV-2 and PMMoV targets, but inhibition appeared to be mitigated by dilution of template RNA. The concentrations of N1 and PMMoV from all three methods were significantly correlated (Pearson's r=0.97-0.98 for N1 and r=0.89-0.93 for PMMoV), although RT-qPCR reported higher concentrations than digital methods. Taken together, this study provides support for the application of all three methods in wastewater-based epidemiology, with additional guidelines for the use of RT-qPCR.

Operationalizing a routine wastewater monitoring laboratory for SARS-CoV-2 (preprint)

Wastewater-based testing for SARS-CoV-2 is a novel tool for public health monitoring, but additional laboratory capacity is needed to provide routine monitoring at all locations where it has the potential to be useful. Few standardization practices for SARS-CoV-2 wastewater analysis currently exist, and quality assurance/quality control procedures may vary across laboratories. Alongside counterparts at many academic institutions, we built out a laboratory for routine monitoring of wastewater at the University of California, Berkeley. Here, we detail our group's establishment of a wastewater testing laboratory including standard operating procedures, laboratory buildout and workflow, and a quality assurance plan. We present a complete data analysis pipeline and quality scoring framework and discuss the data reporting process. We hope that this information will aid others at research institutions, public health departments, and wastewater agencies in developing programs to support wastewater monitoring for public health decision-making.

Interpretation of temporal and spatial trends of SARS-CoV-2 RNA in San Francisco Bay Area wastewater (preprint)

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be integrated with COVID-19 case data to inform timely pandemic response. However, more research is needed to apply and develop systematic methods to interpret the true SARS-CoV-2 signal from noise introduced in wastewater samples (e.g., from sewer conditions, sampling and extraction methods, etc.). In this study, raw wastewater was collected weekly from five sewersheds and one residential facility, and wastewater SARS-CoV-2 concentrations were compared to geocoded COVID-19 clinical testing data. SARS-CoV-2 was reliably detected (95% positivity) in frozen wastewater samples when reported daily new COVID-19 cases were 2.4 or more per 100,000 people. To adjust for variation in sample fecal content, crAssphage, pepper mild mottle virus, Bacteroides ribosomal RNA (rRNA), and human 18S rRNA were evaluated as normalization biomarkers, and crAssphage displayed the least spatial and temporal variability. Both unnormalized SARS-CoV-2 RNA signal and signal normalized to crAssphage had positive and significant correlation with clinical testing data (Kendall's Tau-b=0.43 and 0.38, respectively). Locational dependencies and the date associated with testing data impacted the lead time of wastewater for clinical trends, and no lead time was observed when the sample collection date (versus the result date) was used for both wastewater and clinical testing data. This study supports that trends in wastewater surveillance data reflect trends in COVID-19 disease occurrence and presents approaches that could be applied to make wastewater signal more interpretable and comparable across studies.

Sewage, Salt, Silica and SARS-CoV-2 (4S): An economical kit-free method for direct capture of SARS-CoV-2 RNA from wastewater. (preprint)

Wastewater-based epidemiology is an emerging tool to monitor COVID-19 infection levels by measuring the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. There remains a need to improve wastewater RNA extraction methods sensitivity, speed, and reduce reliance on often expensive commercial reagents to make wastewater-based epidemiology more accessible. We present a kit-free wastewater RNA extraction method, titled "Sewage, Salt, Silica and SARS-CoV-2" (4S), that employs the abundant and affordable reagents sodium chloride (NaCl), ethanol and silica RNA capture matrices to recover 6-fold more SARS-CoV-2 RNA from wastewater than an existing ultrafiltration-based method. The 4S method concurrently recovered pepper mild mottle virus (PMMoV) and human 18S ribosomal subunit rRNA, both suitable as fecal concentration controls. The SARS-CoV-2 RNA concentrations measured in three sewersheds corresponded to the relative prevalence of COVID-19 infection determined via clinical testing. Lastly, controlled experiments indicate that the 4S method prevented RNA degradation during storage of wastewater samples, was compatible with heat pasteurization, and could be performed in approximately 3 hours. Overall, the 4S method is promising for effective, economical, and accessible wastewater-based epidemiology for SARS-CoV-2, providing another tool to fight the global pandemic. SYNOPSISThe 4S method for measuring SARS-CoV-2 in wastewater is promising for effective, economical, and accessible wastewater-based epidemiology. ABSTRACT ART O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/20242131v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@192784eorg.highwire.dtl.DTLVardef@11879e7org.highwire.dtl.DTLVardef@1eb315borg.highwire.dtl.DTLVardef@1f560cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Genome sequencing of sewage detects regionally prevalent SARS-CoV-2 variants (preprint)

Viral genome sequencing has guided our understanding of the spread and extent of genetic diversity of SARS-CoV-2 during the COVID-19 pandemic. SARS-CoV-2 viral genomes are usually sequenced from nasopharyngeal swabs of individual patients to track viral spread. Recently, RT-qPCR of municipal wastewater has been used to quantify the abundance of SARS-CoV-2 in several regions globally. However, metatranscriptomic sequencing of wastewater can be used to profile the viral genetic diversity across infected communities. Here, we sequenced RNA directly from sewage collected by municipal utility districts in the San Francisco Bay Area to generate complete and near-complete SARS-CoV-2 genomes. The major consensus SARS-CoV-2 genotypes detected in the sewage were identical to clinical genomes from the region. Using a pipeline for single nucleotide variant (SNV) calling in a metagenomic context, we characterized minor SARS-CoV-2 alleles in the wastewater and detected viral genotypes which were also found within clinical genomes throughout California. Observed wastewater variants were more similar to local California patient-derived genotypes than they were to those from other regions within the US or globally. Additional variants detected in wastewater have only been identified in genomes from patients sampled outside of CA, indicating that wastewater sequencing can provide evidence for recent introductions of viral lineages before they are detected by local clinical sequencing. These results demonstrate that epidemiological surveillance through wastewater sequencing can aid in tracking exact viral strains in an epidemic context.