Challenges in Measuring the Recovery of SARS-CoV-2 from Wastewater.

Wastewater-based epidemiology is an emerging tool for tracking the spread of SARS-CoV-2 through populations. However, many factors influence recovery and quantification of SARS-CoV-2 from wastewater, complicating data interpretation. Specifically, these factors may differentially affect the measured virus concentration, depending on the laboratory methods used to perform the test. Many laboratories add a proxy virus to wastewater samples to determine losses associated with concentration and extraction of viral RNA. While measuring recovery of a proxy virus is an important process control, in this piece, we describe the caveats and limitations to the interpretation of this control, including that it typically does not account for losses during RNA extraction. We recommend reporting the directly measured concentration data alongside the measured recovery efficiency, rather than attempting to correct the concentration for recovery efficiency. Even though the ability to directly compare SARS-CoV-2 concentrations from different sampling locations determined using different methods is limited, concentration data (uncorrected for recovery) can be useful for public health response.

Sewage, Salt, Silica, and SARS-CoV-2 (4S): An Economical Kit-Free Method for Direct Capture of SARS-CoV-2 RNA from Wastewater.

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 sixfold 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, which have been proposed 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 in our experience, 20 samples can be processed by one lab technician in approximately 2 h. 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.

Microbial Water Quality through a Full-Scale Advanced Wastewater Treatment Demonstration Facility.

The fates of viruses, bacteria, and antibiotic resistance genes during advanced wastewater treatment are important to assess for implementation of potable reuse systems. Here, a full-scale advanced wastewater treatment demonstration facility (ozone, biological activated carbon filtration, micro/ultrafiltration, reverse osmosis, and advanced oxidation) was sampled over three months. Atypically, no disinfectant residual was applied before the microfiltration step. Microbial cell concentrations and viability were assessed via flow cytometry and adenosine triphosphate (ATP). Concentrations of bacteria (16S rRNA gene), viruses (human adenovirus and JC polyomavirus), and antibiotic resistance genes (sul1 and bla TEM ) were assessed via quantitative PCR following the concentration of large sample volumes by dead-end ultrafiltration. In all membrane filtration permeates, microbial concentrations were higher than previously reported for chloraminated membranes, and log10 reduction values were lower than expected. Concentrations of 16S rRNA and sul1 genes were reduced by treatment but remained quantifiable in reverse osmosis permeate. It is unclear whether sul1 in the RO permeate was from the passage of resistance genes or new growth of microorganisms, but the concentrations were on the low end of those reported for conventional drinking water distribution systems. Adenovirus, JC polyomavirus, and bla TEM genes were reduced below the limit of detection (∼10-2 gene copies per mL) by microfiltration. The results provide insights into how treatment train design and operation choices affect microbial water quality as well as the use of flow cytometry and ATP for online monitoring and process control.

Genome Sequencing of Sewage Detects Regionally Prevalent SARS-CoV-2 Variants.

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 nearly 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 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 United States or globally. Additional variants detected in wastewater have only been identified in genomes from patients sampled outside California, 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.

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

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. The concentrations of SARS-CoV-2 in wastewater samples 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, four normalization biomarkers were evaluated: crAssphage, pepper mild mottle virus, Bacteroides ribosomal RNA (rRNA), and human 18S rRNA. Of these, 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 (τ)=0.43 and 0.38, respectively), but no normalization biomarker strengthened the correlation with clinical testing data. 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 tools 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.

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.