Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater.

Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.

Considerations and Opportunities for Probe Capture Enrichment Sequencing of Emerging Viruses from Wastewater.

Until recently, wastewater-based monitoring for pathogens of public health concern primarily used PCR-based quantification methods and targeted sequencing for specific pathogens (e.g., SARS-CoV-2). In the past three years, researchers have expanded sequencing to monitor a broad range of pathogens, applying probe capture enrichment to wastewater. The goals of those studies included (1) monitoring and expanding fundamental knowledge of disease dynamics for known pathogens and (2) evaluating the potential for early detection of emerging diseases resulting from zoonotic spillover or novel viral variants. Several studies using off-the-shelf probe panels designed for clinical and environmental surveillance reported that enrichment increased virus relative abundance but did not recover complete genomes for most nonenteric viruses. Based on our experience and recent results reported by others using these panels for wastewater, clinical, and synthetic samples, we discuss challenges and technical factors that affect the rates of false positive and false negative results. We identify trade-offs and opportunities throughout the workflow, including in wastewater sample processing, probe panel design, and bioinformatic analysis. We suggest tailored methods of virus concentration and background removal, carefully designed probe panels, and multithresholded bioinformatics analysis.

Responses of drinking water bulk and biofilm microbiota to elevated water age in bench-scale simulated distribution systems.

Reductions in nonresidential water demand during the COVID-19 pandemic highlighted the importance of understanding how water age impacts drinking water quality and microbiota in piped distribution systems. Using benchtop model distribution systems, we aimed to characterize the impacts of elevated water age on microbiota in bulk water and pipe wall biofilms. Five replicate constant-flow reactors were fed with municipal chloraminated tap water for 6 months prior to building closures and 7 months after. After building closures, chloramine levels entering the reactors dropped; in the reactor bulk water and biofilms the mean cell counts and ATP concentrations increased over an order of magnitude while the detection of opportunistic pathogens remained low. Water age, and the corresponding physicochemical changes, strongly influenced microbial abundance and community composition. Differential initial microbial colonization also had a lasting influence on microbial communities in each reactor (i.e., historical contingency).

Correlation between wastewater and COVID-19 case incidence rates in major California sewersheds across three variant periods.

Monitoring for COVID-19 through wastewater has been used for adjunctive public health surveillance, with SARS-CoV-2 viral concentrations in wastewater correlating with incident cases in the same sewershed. However, the generalizability of these findings across sewersheds, laboratory methods, and time periods with changing variants and underlying population immunity has not been well described. The California Department of Public Health partnered with six wastewater treatment plants starting in January 2021 to monitor wastewater for SARS-CoV-2, with analyses performed at four laboratories. Using reported PCR-confirmed COVID-19 cases within each sewershed, the relationship between case incidence rates and wastewater concentrations collected over 14 months was evaluated using Spearman's correlation and linear regression. Strong correlations were observed when wastewater concentrations and incidence rates were averaged (10- and 7-day moving window for wastewater and cases, respectively, ρ = 0.73-0.98 for N1 gene target). Correlations remained strong across three time periods with distinct circulating variants and vaccination rates (winter 2020-2021/Alpha, summer 2021/Delta, and winter 2021-2022/Omicron). Linear regression revealed that slopes of associations varied by the dominant variant of concern, sewershed, and laboratory (ß = 0.45-1.94). These findings support wastewater surveillance as an adjunctive public health tool to monitor SARS-CoV-2 community trends.

O2 partitioning of sulfur oxidizing bacteria drives acidity and thiosulfate distributions in mining waters.

The acidification of water in mining areas is a global environmental issue primarily catalyzed by sulfur-oxidizing bacteria (SOB). Little is known about microbial sulfur cycling in circumneutral pH mine tailing impoundment waters. Here we investigate biological sulfur oxidation over four years in a mine tailings impoundment water cap, integrating aqueous sulfur geochemistry, genome-resolved metagenomics and metatranscriptomics. The microbial community is consistently dominated by neutrophilic, chemolithoautotrophic SOB (relative abundances of ~76% in 2015, ~55% in 2016/2017 and ~60% in 2018). Results reveal two SOB strategies alternately dominate across the four years, influencing acid generation and sulfur speciation. Under oxic conditions, novel Halothiobacillus drive lower pH conditions (as low as 4.3) and lower [S2O32-] via the complete Sox pathway coupled to O2. Under anoxic conditions, Thiobacillus spp. dominate in activity, via the incomplete Sox and rDSR pathways coupled to NO3-, resulting in higher [S2O32-] and no net significant acidity generation. This study provides genomic evidence explaining acidity generation and thiosulfate accumulation patterns in a circumneutral mine tailing impoundment and has significant environmental applications in preventing the discharge of sulfur compounds that can impact downstream environments. These insights illuminate opportunities for in situ biotreatment of reduced sulfur compounds and prediction of acidification events using gene-based monitoring and in situ RNA detection.

The dynamic relationship between COVID-19 cases and SARS-CoV-2 wastewater concentrations across time and space: Considerations for model training data sets.

During the COVID-19 pandemic, wastewater-based surveillance has been used alongside diagnostic testing to monitor infection rates. With the decline in cases reported to public health departments due to at-home testing, wastewater data may serve as the primary input for epidemiological models, but training these models is not straightforward. We explored factors affecting noise and bias in the ratio between wastewater and case data collected in 26 sewersheds in California from October 2020 to March 2022. The strength of the relationship between wastewater and case data appeared dependent on sampling frequency and population size, but was not increased by wastewater normalization to flow rate or case count normalization to testing rates. Additionally, the lead and lag times between wastewater and case data varied over time and space, and the ratio of log-transformed individual cases to wastewater concentrations changed over time. This ratio decreased between the Epsilon/Alpha and Delta variant surges of COVID-19 and increased during the Omicron BA.1 variant surge, and was also related to the diagnostic testing rate. Based on this analysis, we present a framework of scenarios describing the dynamics of the case to wastewater ratio to aid in data handling decisions for ongoing modeling efforts.

Wastewater Surveillance to Inform Public Health Decision Making in Residential Institutions.

Testing sewage (wastewater-based surveillance, or WBS) for pathogens is an increasingly important tool for monitoring the health of populations. During the COVID-19 pandemic, some residential institutions including colleges, prisons, and skilled nursing facilities used facility-level wastewater data to inform their pandemic responses. To understand how these early adopters used WBS data in decision making, we conducted in-depth, semistructured interviews with multiple decision makers at 6 residential institutions in the United States (universities, prisons, and nursing homes) encompassing a total of more than 70 000 residents and staff about interpretation, uses, and limitations of these data. We found that WBS data were used in extremely diverse ways. WBS combined with clinical surveillance informed a wide range of public health actions at residential institutions, including transmission reduction measures, public health communications, and allocation of resources. WBS also served other institutional purposes, such as maintaining relationships with external stakeholders and helping alleviate decision makers' pervasive stress. Recognizing these diverse ways of using WBS data can inform expansion of this practice among institutions as well as development of community-scale systems.

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.

Genetic diversity and evolutionary convergence of cryptic SARS- CoV-2 lineages detected via wastewater sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population.

Genetic Diversity and Evolutionary Convergence of Cryptic SARS-CoV-2 Lineages Detected Via Wastewater Sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitution. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from immunocompromised patients or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population. Author Summary: During the COVID-19 pandemic, wastewater-based epidemiology has become an effective public health tool. Because many infected individuals shed SARS-CoV-2 in feces, wastewater has been monitored to reveal infection trends in the sewersheds from which the samples were derived. Here we report novel SARS-CoV-2 lineages in wastewater samples obtained from 3 different states in the USA. These lineages appeared in specific sewersheds intermittently over periods of up to 14 months, but generally have not been detected beyond the sewersheds in which they were initially found. Many of these lineages may have diverged in early 2020. Although these lineages share considerable overlap with each other, they have never been observed in patients anywhere in the world. While the wastewater lineages have similarities with lineages observed in long-term infections of immunocompromised patients, animal reservoirs cannot be ruled out as a potential source.