Quantitative analysis of SARS-CoV-2 RNA from wastewater solids in communities with low COVID-19 incidence and prevalence.

Wastewater-based epidemiology may be useful for informing public health response to viral diseases like COVID-19 caused by SARS-CoV-2. We quantified SARS-CoV-2 RNA in wastewater influent and primary settled solids in two wastewater treatment plants to inform the preanalytical and analytical approaches and to assess whether influent or solids harbored more viral targets. The primary settled solids samples resulted in higher SARS-CoV-2 detection frequencies than the corresponding influent samples. Likewise, SARS-CoV-2 RNA was more readily detected in solids using one-step digital droplet (dd)RT-PCR than with two-step RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal time series of 89 settled solids samples from a single plant for SARS-CoV-2 RNA as well as coronavirus recovery (bovine coronavirus) and fecal strength (pepper mild mottle virus) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlated positively and significantly with COVID-19 clinically confirmed case counts in the sewershed. Together, the results demonstrate that measuring SARS-CoV-2 RNA concentrations in settled solids may be a more sensitive approach than measuring SARS-CoV-2 in influent.

SARS-CoV-2 RNA in Wastewater Settled Solids Is Associated with COVID-19 Cases in a Large Urban Sewershed.

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.

/pdf/genome-sequencing-of-sewage-detects-regionally-prevalent-59jr9k5lux.pdf

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

In response to COVID-19, the international water community rapidly developed methods to quantify the SARS-CoV-2 genetic signal in untreated wastewater. Wastewater surveillance using such methods has the potential to complement clinical testing in assessing community health. This interlaboratory assessment evaluated the reproducibility and sensitivity of 36 standard operating procedures (SOPs), divided into eight method groups based on sample concentration approach and whether solids were removed. Two raw wastewater samples were collected in August 2020, amended with a matrix spike (betacoronavirus OC43), and distributed to 32 laboratories across the U.S. Replicate samples analyzed in accordance with the project's quality assurance plan showed high reproducibility across the 36 SOPs: 80% of the recovery-corrected results fell within a band of ±1.15 log10 genome copies per L with higher reproducibility observed within a single SOP (standard deviation of 0.13 log10). The inclusion of a solids removal step and the selection of a concentration method did not show a clear, systematic impact on the recovery-corrected results. Other methodological variations (e.g., pasteurization, primer set selection, and use of RT-qPCR or RT-dPCR platforms) generally resulted in small differences compared to other sources of variability. These findings suggest that a variety of methods are capable of producing reproducible results, though the same SOP or laboratory should be selected to track SARS-CoV-2 trends at a given facility. The methods showed a 7 log10 range of recovery efficiency and limit of detection highlighting the importance of recovery correction and the need to consider method sensitivity when selecting methods for wastewater surveillance.

/pdf/reproducibility-and-sensitivity-of-36-methods-to-quantify-spj0ozrtop.pdf

Reproducibility and sensitivity of 36 methods to quantify the SARS-CoV-2 genetic signal in raw wastewater: findings from an interlaboratory methods evaluation in the U.S.

The World Health Organization (WHO) classified COVID-19 as a global pandemic, with the situation ultimately requiring unprecedented measures to mitigate the effects on public health and the global economy. Although SARS-CoV-2 (the virus responsible for COVID-19) is primarily respiratory in nature, multiple studies confirmed its genetic material could be detected in the feces of infected individuals, thereby highlighting sewage as a potential indicator of community incidence or prevalence. Numerous wastewater surveillance studies subsequently confirmed detection of SARS-CoV-2 RNA in wastewater and wastewater-associated solids/sludge. However, the methods employed in early studies vary widely so it is unclear whether differences in reported concentrations reflect true differences in epidemiological conditions, or are instead driven by methodological artifacts. The current study aimed to compare the performance of virus recovery and detection methods, detect and quantify SARS-CoV-2 genetic material in two Southern Nevada sewersheds from March–May 2020, and better understand the potential link between COVID-19 incidence/prevalence and wastewater concentrations of SARS-CoV-2 RNA. SARS-CoV-2 surrogate recovery (0.34%–55%) and equivalent sample volume (0.1 mL–1 L) differed between methods and target water matrices, ultimately impacting method sensitivity and reported concentrations. Composite sampling of influent and primary effluent resulted in a ∼10-fold increase in concentration relative to corresponding grab primary effluent samples, presumably highlighting diurnal variability in SARS-CoV-2 signal. Detection and quantification of four SARS-CoV-2 genetic markers (up to ∼106 gene copies per liter), along with ratios of SARS-CoV-2 to pepper mild mottle virus (PMMoV), exhibited comparability with public health data for two sewersheds in an early phase of the pandemic. Finally, a wastewater model informed by fecal shedding rates highlighted the potential significance of new cases (i.e., incidence rather than prevalence) when interpreting wastewater surveillance data.

Early-pandemic wastewater surveillance of SARS-CoV-2 in Southern Nevada: Methodology, occurrence, and incidence/prevalence considerations

COVID-19 surveillance in Southeastern Virginia using wastewater-based epidemiology.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires a significant, coordinated public health response. Assessing case density and spread of infection is critical and relies largely on clinical testing data. However, clinical testing suffers from known limitations, including test availability and a bias towards enumerating only symptomatic individuals. Wastewater-based epidemiology (WBE) has gained widespread support as a potential complement to clinical testing for assessing COVID-19 infections at the community scale. The efficacy of WBE hinges on the ability to accurately characterize SARS-CoV-2 concentrations in wastewater. To date, a variety of sampling schemes have been used without consensus around the appropriateness of grab or composite sampling. Here we address a key WBE knowledge gap by examining the variability of SARS-CoV-2 concentrations in wastewater grab samples collected every 2 hours for 72 hours compared with corresponding 24-hour flow-weighted composite samples. Results show relatively low variability (mean for all assays = 741 copies 100 mL-1, standard deviation = 508 copies 100 mL-1) for grab sample concentrations, and good agreement between most grab samples and their respective composite (mean deviation from composite = 159 copies 100 mL-1). When SARS-CoV-2 concentrations are used to calculate viral load, the discrepancy between grabs (log10 difference = 12.0) or a grab and its associated composite (log10 difference = 11.8) are amplified. A similar effect is seen when estimating carrier prevalence in a catchment population with median estimates based on grabs ranging 62-1853 carriers. Findings suggest that grab samples may be sufficient to characterize SARS-CoV-2 concentrations, but additional calculations using these data may be sensitive to grab sample variability and warrant the use of flow-weighted composite sampling. These data inform future WBE work by helping determine the most appropriate sampling scheme and facilitate sharing of datasets between studies via consistent methodology.

/pdf/wastewater-sars-cov-2-concentration-and-loading-variability-1xwuvk8pf0.pdf

Wastewater SARS-CoV-2 Concentration and Loading Variability from Grab and 24-Hour Composite Samples

Collection system investigation microbial source tracking (CSI-MST): Applying molecular markers to identify sewer infrastructure failures.

The use of ozone/biofiltration advanced treatment has become more prevalent in recent years, with many utilities seeking an alternative to membrane/RO based treatment for water reuse. Ensuring efficient pathogen reduction while controlling disinfection byproducts and maximizing oxidation of trace organic contaminants remains a major barrier to implementing ozone in reuse applications. Navigating these challenges is imperative in order to allow for the more widespread application of ozonation. Here, we demonstrate the effectiveness of ozone for virus, coliform bacteria, and spore forming bacteria inactivation in unfiltered secondary effluent, all the while controlling the disinfection byproduct bromate. A greater than 6-log reduction of both male specific and somatic coliphages was seen at specific ozone doses as low as 0.75 O3:TOC. This study compared monochloramine and hydrogen peroxide as chemical bromate control measures in high bromide water (Br- = 0.35 ± 0.07 mg/L). On average, monochloramine and hydrogen peroxide resulted in an 80% and 36% decrease of bromate formation, respectively. Neither bromate control method had any appreciable impact on virus or coliform bacteria disinfection by ozone; however, the use of hydrogen peroxide would require a non-Ct disinfection framework. Maintaining ozone residual was shown to be critical for achieving disinfection of more resilient microorganisms, such as spore forming bacteria. While extremely effective at controlling bromate, monochloramine was shown to inhibit TrOC oxidation, whereas hydrogen peroxide enhanced TrOC oxidation.

Kathleen Yetka

Papers

COVID-19 surveillance in Southeastern Virginia using wastewater-based epidemiology.

Wastewater SARS-CoV-2 Concentration and Loading Variability from Grab and 24-Hour Composite Samples

Collection system investigation microbial source tracking (CSI-MST): Applying molecular markers to identify sewer infrastructure failures.

Optimizing Ozone Disinfection in Water Reuse: Controlling Bromate Formation and Enhancing Trace Organic Contaminant Oxidation.