Structured Ethical Review for Wastewater-Based Testing in Support of Public Health.

Wastewater-based testing (WBT) for SARS-CoV-2 has rapidly expanded over the past three years due to its ability to provide a comprehensive measurement of disease prevalence independent of clinical testing. The development and simultaneous application of WBT measured biomarkers for research activities and for the pursuit of public health goals, both areas with well-established ethical frameworks. Currently, WBT practitioners do not employ a standardized ethical review process, introducing the potential for adverse outcomes for WBT professionals and community members. To address this deficiency, an interdisciplinary workshop developed a framework for a structured ethical review of WBT. The workshop employed a consensus approach to create this framework as a set of 11 questions derived from primarily public health guidance. This study retrospectively applied these questions to SARS-CoV-2 monitoring programs covering the emergent phase of the pandemic (3/2020-2/2022 (n = 53)). Of note, 43% of answers highlight a lack of reported information to assess. Therefore, a systematic framework would at a minimum structure the communication of ethical considerations for applications of WBT. Consistent application of an ethical review will also assist in developing a practice of updating approaches and techniques to reflect the concerns held by both those practicing and those being monitored by WBT supported programs.

Review: Abandoned mines as a resource or liability for wildlife.

Abandoned Mine Lands (AMLs) are areas where previous mineral extraction or processing has occurred. Hundreds of thousands of AMLs exist within the United States. Contaminated runoff from AMLs can negatively affect the physiology and ecology of surrounding terrestrial and aquatic habitats and species and can be detrimental to human health. As a response, several U.S. federal and state agencies have launched programs to assess health risks associated with AMLs. In some cases, however, AMLs may be beneficial to specific wildlife taxa. There is a relative paucity of studies investigating the physiological and ecological impacts of AMLs on wildlife. We conducted a systematic review examining published scientific articles that assessed the negative and positive impacts of AMLs across invertebrate and vertebrate taxa. We also offer suggestions on evaluating AMLs to develop effective mitigation strategies that reduce their negative tole on human and wildlife communities. Peer-reviewed publications were screened across WebofScience, PubMed and Google Scholar databases. Abandoned mine lands were generally detrimental to wildlife, with adverse effects ranging from bioaccumulation of heavy metals to decreased ecological fitness. Conversely, AMLs were an overall benefit to imperiled bat populations and could serve as tools for conservation. Studies were unevenly distributed across different wildlife taxa groups, echoing the necessity for additional taxonomically diverse research. We suggest that standardized wildlife survey methods be used to assess how different species utilize AMLs. Federal and state agencies can use these surveys to establish effective remediation plans for individual AML sites and minimize the risks to both wildlife and humans.

Carcinogenic formaldehyde in U.S. residential buildings: Mass inventories, human health impacts, and associated healthcare costs.

Formaldehyde, a human carcinogen, is formulated into building materials in the U.S. and worldwide. We used literature information and mass balances to obtain order-of-magnitude estimates of formaldehyde inventories in U.S. residential buildings as well as associated exposures, excess morbidity, and healthcare costs along with other economic ramifications. Use of formaldehyde in building materials dates to the 1940s and continues today unabated, despite its international classification in 2004 as a human carcinogen. Global production of formaldehyde was about 32 million metric tons (MMT) in 2006. In the U.S., 5.7 ± 0.05 to 7.4 ± 0.125 MMT of formaldehyde were produced annually from 2006 to 2022, with 65 ± 5 % of this mass (3.7 ± 0.03 to 4.8 ± 0.08 MMT) entering building materials. For a typical U.S. residential building constructed in 2022, we determined an average total mass of formaldehyde containing chemicals of 48.2 ± 10.1 kg, equivalent to 207 ± 40 g of neat formaldehyde per housing unit. When extrapolated to the entire U.S. housing stock, this equates to 29,800 ± 5760 metric tons of neat formaldehyde. If the health threshold in indoor air of 0.1 mg/m3 is never surpassed in a residential building, safe venting of embedded formaldehyde would take years. Using reported indoor air exceedances, up to 645 ± 33 excess cancer cases may occur U.S. nationwide annually generating up to US$65 M in cancer treatment costs alone, not counting ~16,000 ± 1000 disability adjusted life-years. Other documents showed health effects of formaldehyde exist, but could not be quantified reliably, including sick building syndrome outcomes such as headache, asthma, and various respiratory illnesses. Opportunities to improve indoor air exposure assessments are discussed with special emphasis on monitoring of building wastewater. Safer alternatives to formaldehyde in building products exist and are recommended for future use.

Encrypted data-sharing for preserving privacy in wastewater-based epidemiology.

The rapidly expanding use of wastewater for public health surveillance requires new strategies to protect privacy rights, while data are collected at increasingly discrete geospatial scales, i.e., city, neighborhood, campus, and building-level. Data collected at high geospatial resolution can inform on labile, short-lived biomarkers, thereby making wastewater-derived data both more actionable and more likely to cause privacy concerns and stigmatization of subpopulations. Additionally, data sharing restrictions among neighboring cities and communities can complicate efforts to balance public health protections with citizens' privacy. Here, we have created an encrypted framework that facilitates the sharing of sensitive population health data among entities that lack trust for one another (e.g., between adjacent municipalities with different governance of health monitoring and data sharing). We demonstrate the utility of this approach with two real-world cases. Our results show the feasibility of sharing encrypted data between two municipalities and a laboratory, while performing secure private computations for wastewater-based epidemiology (WBE) with high precision, fast speeds, and low data costs. This framework is amenable to other computations used by WBE researchers including population normalized mass loads, fecal indicator normalizations, and quality control measures. The Centers for Disease Control and Prevention's National Wastewater Surveillance System shows ∼8 % of the records attributed to collection before the wastewater treatment plant, illustrating an opportunity to further expand currently limited community-level sampling and public health surveillance through security and responsible data-sharing as outlined here.

Seasonality of respiratory, enteric, and urinary viruses revealed by wastewater genomic surveillance.

Wastewater surveillance can reveal population-level infectious disease burden and emergent public health threats can be reliably assessed through wastewater surveillance. While molecular methods for wastewater monitoring of microorganisms have traditionally relied on PCR-based approaches, next-generation sequencing (NGS) can provide deeper insights via genomic analyses of multiple diverse pathogens. We conducted a year-long sequencing surveillance of 1,408 composite wastewater samples collected from 12 neighborhood-level access points in the greater Tempe area, Arizona, USA, and show that variation in wastewater viruses is driven by seasonal time and location. The temporal dynamics of viruses in wastewater were influenced cyclically, with the most dissimilarity between samples 23 weeks apart (i.e., winter vs summer, spring vs fall). We identified diverse urinary and enteric viruses including polyomaviruses, astroviruses, and noroviruses, and showed that their genotypes/subtypes shifted across seasons. We show that while wastewater data of certain respiratory viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strongly correlate with clinical case rates, laboratory-reported case incidences were discordant with surges of high viral load in wastewater for other viruses like human coronavirus 229E. These results demonstrate the utility of wastewater sequencing for informing decision-making in public health.IMPORTANCEWastewater surveillance can provide insights into the spread of pathogens in communities. Advances in next-generation sequencing (NGS) methodologies allow for more precise detection of viruses in wastewater. Long-term wastewater surveillance of viruses is an important tool for public health preparedness. This system can act as a public health observatory that gives real-time early warning for infectious disease outbreaks and improved response times.

Direct detection of canine picornavirus complete coding sequence in wastewater using long-range reverse-transcriptase polymerase chain reaction and long-read sequencing.

We describe four complete coding sequence (cCDS) of canine picornavirus from wastewater in Arizona, USA detected by coupling cCDS single-contig (∼7.5 kb) reverse-transcriptase polymerase chain reaction (RT-PCR) and low-cost long-read high-throughput sequencing. For viruses of medical/veterinary importance, this workflow expands possibilities of wastewater based genomic epidemiology for exploring virus evolutionary dynamics especially in low-resource settings.

Wastewater and clinical surveillance of respiratory viral pathogens on a university campus.

Areas of dense population congregation are prone to experience respiratory virus outbreaks. We monitored wastewater and clinic patients for the presence of respiratory viruses on a large, public university campus. Campus sewer systems were monitored in 16 locations for the presence of viruses using next generation sequencing over 22 weeks in 2023. During this period, we detected a surge in human adenovirus (HAdV) levels in wastewater. Hence, we initiated clinical surveillance at an on-campus clinic from patients presenting with acute respiratory infection. From whole genome sequencing of 123 throat and/or nasal swabs collected, we identified an outbreak of HAdV, specifically of HAdV-E4 and HAdV-B7 genotypes overlapping in time. The temporal dynamics and proportions of HAdV genotypes found in wastewater were corroborated in clinical infections. We tracked specific single nucleotide polymorphisms (SNPs) found in clinical virus sequences and showed that they arose in wastewater signals concordant with the time of clinical presentation, linking community transmission of HAdV to the outbreak. This study demonstrates how wastewater-based epidemiology can be integrated with surveillance at ambulatory healthcare settings to monitor areas prone to respiratory virus outbreaks and provide public health guidance.

Determining the connectivity of tribal communities to wastewater treatment facilities for use in environmental contamination and exposure assessments by wastewater-based surveillance.

BACKGROUND: Limited information is available on the connectivity of Tribal communities to wastewater treatment facilities (WWTFs). This is important for understanding current sanitation infrastructure which drives public health and community construction, knowledge of potential routes of exposure through lack of infrastructure and/or discharging facilities, and opportunities to assess community health through wastewater-based surveillance (WBS). OBJECTIVES: The objective of this work was to assess current wastewater infrastructure for 574 Federally Recognized Indian Tribes (FRITs) in the United States (US) to determine the number and location of facilities on or adjacent to Tribal reservations and Off-Reservation Trust Lands, with the goal of determining the feasibility of employing wastewater-based surveillance within these communities and to identify areas with inadequate sanitation infrastructure. METHODS: Here, we identified available National Pollutant Discharge Elimination System (NPDES) wastewater discharge permits in the Environmental Protection Agency's Environmental Compliance History Online database to assess proximity to and within spatial boundaries of Tribal lands. These data were coupled to race data and tribal spatial boundary information from the US Census Bureau. RESULTS: 94 FRITs have registered NPDES permits within Tribal boundaries including a total of 522 facilities. 210 of these are American Indian (AI)-serving (>50% AI) with the ability to reach 135,000 AI-people through the wastewater network to provide community health assessments via WBS. Of the remaining facilities, 153 predominantly serve non-Tribal populations raising concerns about infrastructure placement and indigenous sovereignty. 523 FRITs were identified as without permitted discharging WWTFs, which may suggest inadequate or alternative infrastructure. IMPACT STATEMENT: Here, multiple data sources including permit information from the Environmental Protection Agency's National Pollution Discharge Elimination System and US Census Bureau data were used to determine the number of wastewater treatment facilities on or adjacent to Tribal lands and how many community members were connected to those municipal systems. This information was used to assess which Tribal communities may be a viable option for wastewater public health surveillance techniques and were used to answer supplemental questions related to basic sanitation and environmental justice concerns.

Nine Rhizobium phage genomes recovered from wastewater in Tempe, AZ, October 2019-March 2020.

We describe nine Rhizobium microvirus genomes identified in wastewater in Tempe, AZ, USA, between October 2019 and March 2020. The major capsid protein (MCP) encoded in these genomes phylogenetically cluster together and are distinct from the MCPs of Rhizobium microviruses identified in Mexico and Argentina.

Human adenovirus outbreak at a university campus monitored by wastewater and clinical surveillance.

Areas of dense population congregation are prone to experience respiratory virus outbreaks. We monitored wastewater and clinic patients for the presence of respiratory viruses on a large, public university campus. Campus sewer systems were monitored in 16 locations for the presence of viruses using next generation sequencing over 22 weeks in 2023. During this period, we detected a surge in human adenovirus (HAdV) levels in wastewater. Hence, we initiated clinical surveillance at an on-campus clinic from patients presenting with acute respiratory infection. From whole genome sequencing of 123 throat and/or nasal swabs collected, we identified an outbreak of HAdV, specifically of HAdV-E4 and HAdV-B7 genotypes overlapping in time. The temporal dynamics and proportions of HAdV genotypes found in wastewater were corroborated in clinical infections. We tracked specific single nucleotide polymorphisms (SNPs) found in clinical virus sequences and showed that they arose in wastewater signals concordant with the time of clinical presentation, linking community transmission of HAdV to the outbreak. This study demonstrates how wastewater-based epidemiology can be integrated with surveillance at ambulatory healthcare settings to monitor areas prone to respiratory virus outbreaks and provide public health guidance.

Prevalence rates of neurodegenerative diseases versus human exposures to heavy metals across the United States.

Novel means are needed to identify individuals and subpopulations susceptible to and afflicted by neurodegenerative diseases (NDDs). This study aimed to utilize geographic distribution of heavy metal sources and sinks to investigate a potential human health risk of developing NDDs. Known or hypothesized environmental factors driving disease prevalence of Alzheimer's Disease (AD), Parkinson's Disease (PD), and amyotrophic lateral sclerosis (ALS) are heavy metals, including arsenic (As), cadmium (Cd), manganese (Mn) and mercury (Hg). Lead (Pb) has been associated with AD and ALS. Analyzable mediums of human exposure to heavy metals (i.e., toxic metals and metalloids), or proxies thereof, include infant blood, topsoil, sewage sludge, and well water. U.S. concentrations of heavy metals in topsoil, sewage sludge, well water, and infant blood were mapped and compared to prevalence rates of major NDDs. Data from federal and state agencies (i.e., CDC, EPA, and the US Geological Survey) on heavy metal concentrations, age distribution, and NDD prevalence rates were geographically represented and statistically analyzed to quantify possible correlations. Aside from an expected significant association between NDD prevalence and age (p < 0.0001), we found significant associations between the prevalence of the sum of three major NDDs with: Pb in topsoil (p = 0.0433); Cd (p < 0.0001) and Pb (p < 0.0001) in sewage sludge; Pb in infant blood (p < 0.0001). Concentrations in sewage sludge of Cd and Pb were significantly correlated with NDD prevalence rates with an odds ratio of 2.91 (2.04, 4.225 95%CI) and 4.084 (3.14, 5.312 95%CI), respectively. The presence of toxic metals in the U.S. environment in multiple matrices, including sewage sludge, was found to be significantly associated with NDD prevalence. This is the first use of sewage sludge as an environmental proxy matrix to infer risk of developing NDDs.

Wastewater-based monitoring of the nitazene analogues: First detection of protonitazene in wastewater.

Synthetic opioids, particularly the nitazene analogues class, have become a public health concern due to their high potency. Wastewater-based epidemiology can detect community use of these compounds. The objective of this work was to detect nitazene analogues in wastewater from samples collected from eight sites in the United States. Influent wastewater samples were collected from eight sites in seven states (Arizona, Oregon, New Mexico, Illinois, New Jersey, Washington and Georgia) in the United States. Samples were collected from each site on three days between 27 December 2022 and 4 January 2023, acidified on collection, stored frozen and shipped to Arizona State University (Tempe, AZ) for sample processing. Samples were then shipped to The University of Queensland (Brisbane, Australia) for sample analysis. Protonitazene was found in samples collected from two sites in Washington and Illinois. The concentration was estimated up to 0.5 ng/L, with estimated excreted mass loads up to 0.3 mg/day/1000 people. This work has shown that it is possible to detect nitazene analogues in wastewater using a combination of sample pre-concentration and sensitive instrumentation, thereby further expanding the utility of wastewater-based epidemiology.

Exploring Canine Picornavirus Diversity in the USA Using Wastewater Surveillance: From High-Throughput Genomic Sequencing to Immuno-Informatics and Capsid Structure Modeling.

The SARS-CoV-2 pandemic resulted in a scale-up of viral genomic surveillance globally. However, the wet lab constraints (economic, infrastructural, and personnel) of translating novel virus variant sequence information to meaningful immunological and structural insights that are valuable for the development of broadly acting countermeasures (especially for emerging and re-emerging viruses) remain a challenge in many resource-limited settings. Here, we describe a workflow that couples wastewater surveillance, high-throughput sequencing, phylogenetics, immuno-informatics, and virus capsid structure modeling for the genotype-to-serotype characterization of uncultivated picornavirus sequences identified in wastewater. Specifically, we analyzed canine picornaviruses (CanPVs), which are uncultivated and yet-to-be-assigned members of the family Picornaviridae that cause systemic infections in canines. We analyzed 118 archived (stored at -20 °C) wastewater (WW) samples representing a population of ~700,000 persons in southwest USA between October 2019 to March 2020 and October 2020 to March 2021. Samples were pooled into 12 two-liter volumes by month, partitioned (into filter-trapped solids [FTSs] and filtrates) using 450 nm membrane filters, and subsequently concentrated to 2 mL (1000×) using 10,000 Da MW cutoff centrifugal filters. The 24 concentrates were subjected to RNA extraction, CanPV complete capsid single-contig RT-PCR, Illumina sequencing, phylogenetics, immuno-informatics, and structure prediction. We detected CanPVs in 58.3% (14/24) of the samples generated 13,824,046 trimmed Illumina reads and 27 CanPV contigs. Phylogenetic and pairwise identity analyses showed eight CanPV genotypes (intragenotype divergence <14%) belonging to four clusters, with intracluster divergence of <20%. Similarity analysis, immuno-informatics, and virus protomer and capsid structure prediction suggested that the four clusters were likely distinct serological types, with predicted cluster-distinguishing B-cell epitopes clustered in the northern and southern rims of the canyon surrounding the 5-fold axis of symmetry. Our approach allows forgenotype-to-serotype characterization of uncultivated picornavirus sequences by coupling phylogenetics, immuno-informatics, and virus capsid structure prediction. This consequently bypasses a major wet lab-associated bottleneck, thereby allowing resource-limited settings to leapfrog from wastewater-sourced genomic data to valuable immunological insights necessary for the development of prophylaxis and other mitigation measures.

Inter-institutional laboratory standardization for SARS-CoV-2 surveillance through wastewater-based epidemiology applied to Mexico City.

Objectives: Wastewater-based surveillance applied to SARS-CoV-2 viral load quantification for COVID-19 has become one of the most relevant complementary tools in epidemiologic prevention programs worldwide. However, this valuable decision-making tool still requires fine-tuning to produce comparable results between laboratories, especially when applied to the surveillance of megacities. Methods: Six laboratories across Mexico and one from the United States executed an interlaboratory study to set up a singular standardized protocol considering method cost, installed infrastructure, materials available, and supply availability for SARS-CoV-2 quantification from five Mexico City sampling sites across this megacity. Results: Comparable data from processing outcomes in the Mexican laboratories and in the external international laboratory serve as a validating data source. The Bland-Altman comparison showed consistency, with cycle threshold values within ±1.96 SD of SARS-CoV-2 genetic copies for the standard curve quantification, with a mismatch of two laboratories. In addition, MS2 bacteriophage recovery rates varied between 35% and 67% among all participating laboratories. Finally, the efficiency of viral genetic material recovered from all participating laboratories varied between 65% and 93% for the participating laboratories. Conclusion: This work lays the foundation for extensive and continuous wastewater-based surveillance application across independent Mexican laboratories in a time- and resource-effective manner.

Workflow to facilitate the detection of new psychoactive substances and drugs of abuse in influent urban wastewater.

The complexity around the dynamic markets for new psychoactive substances (NPS) forces researchers to develop and apply innovative analytical strategies to detect and identify them in influent urban wastewater. In this work a comprehensive suspect screening workflow following liquid chromatography - high resolution mass spectrometry analysis was established utilising the open-source InSpectra data processing platform and the HighResNPS library. In total, 278 urban influent wastewater samples from 47 sites in 16 countries were collected to investigate the presence of NPS and other drugs of abuse. A total of 50 compounds were detected in samples from at least one site. Most compounds found were prescription drugs such as gabapentin (detection frequency 79%), codeine (40%) and pregabalin (15%). However, cocaine was the most found illicit drug (83%), in all countries where samples were collected apart from the Republic of Korea and China. Eight NPS were also identified with this protocol: 3-methylmethcathinone 11%), eutylone (6%), etizolam (2%), 3-chloromethcathinone (4%), mitragynine (6%), phenibut (2%), 25I-NBOH (2%) and trimethoxyamphetamine (2%). The latter three have not previously been reported in municipal wastewater samples. The workflow employed allowed the prioritisation of features to be further investigated, reducing processing time and gaining in confidence in their identification.

Stability of human stress hormones and stress hormone metabolites in wastewater under oxic and anoxic conditions.

Levels in wastewater of human stress biomarkers, such as cortisone (E), cortisol (F), tetrahydrocortisone (THE), and tetrahydrocortisol (THF) may serve as indicators of population wellbeing and overall health. This study examined the stability of these biosignature compounds in wastewater to inform on their applicability for use in wastewater-based epidemiology (WBE). Wastewater from two undisclosed U.S. municipalities were fortified with the above four biomarkers of stress to a concentration of 10 ppb, and their decay was studied at three temperatures (15, 25, and 35 °C) over 24 h in oxic and anoxic conditions. Samples were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS) in conjunction with the isotope dilution method for absolute quantitation. Results demonstrated short-term persistence (24 h) of biomarkers at low temperatures (15 °C), and accelerating kinetics of decay that were positively correlated with temperature increases. Among the four biomarkers evaluated, the tetrahydro derivatives were the most long-lived sewage-borne stress biomarkers and these are recommended as prime analytical targets for use in WBE when tracking population stress. Statistical analyses using a non-parametric Wilcoxon test further revealed no significant differences (p > 0.05) between oxic and anoxic decay rates for all stress biomarkers in wastewater from all study locations, regardless of the prevailing temperature regime. This negative finding is worthy of reporting because it suggests the feasibility of straightforward modeling of stress hormone decay, irrespective of whether the sewerage system monitored contains fully filled, pressurized pipes or partially filled gravity flow pipes, whose filling level, and with it its redox conditions, are known to fluctuate over time with water use and storm events.

Enhanced detection of mpox virus in wastewater using a pre-amplification approach: A pilot study informing population-level monitoring of low-titer pathogens.

A recent outbreak of the mpox virus (MPXV) occurred in non-endemic regions of the world beginning in May 2022. Pathogen surveillance systems faced pressure to quickly establish response protocols, offering an opportunity to employ wastewater-based epidemiology (WBE) for population-level monitoring. The pilot study reported herein aimed to: (i) develop a reliable protocol for MPXV DNA detection in wastewater which would reduce false negative reporting, (ii) test this protocol on wastewater from various regions across the United States, and (iii) conduct a state of the science review of the current literature reporting on experimental methods for MPXV detection using WBE. Twenty-four-hour composite samples of untreated municipal wastewater were collected from the states of New Jersey, Georgia, Illinois, Texas, Arizona, and Washington beginning July 3rd, 2022 through October 16th, 2022 (n = 60). Samples underwent vacuum filtration, DNA extraction from captured solids, MPXV DNA pre-amplification, and qPCR analysis. Of the 60 samples analyzed, a total of eight (13%) tested positive for MPXV in the states of Washington, Texas, New Jersey, and Illinois. The presence of clade IIb MPXV DNA in these samples was confirmed via Sanger sequencing and integration of pre-amplification prior to qPCR decreased the rate of false negative detections by 87% as compared to qPCR analysis alone. Wastewater-derived detections of MPXV were compared to clinical datasets, with 50% of detections occurring as clinical cases were increasing/peaking and 50% occurring as clinical cases waned. Results from the literature review (n = 9 studies) revealed successful strategies for the detection of MPXV DNA in wastewater, however also emphasized a need for further method optimization and standardization. Overall, this work highlights the use of pre-amplification prior to qPCR detection as a means to capture the presence of MPXV DNA in community wastewater and offers guidance for monitoring low-titer pathogens via WBE.

Integrated multiomic wastewater-based epidemiology can elucidate population-level dietary behaviour and inform public health nutrition assessments.

Population-level nutritional assessments often rely on self-reported data, which increases the risk of recall bias. Here, we demonstrate that wastewater-based epidemiology can be used for near real-time population dietary assessments. Neighbourhood-level, untreated wastewater samples were collected monthly from within an urban population in the south-western United States from August 2017 to July 2019. Using liquid chromatography-tandem mass spectrometry, we identify recurring seasonal dynamics in phytoestrogen consumption, including dietary changes linked to the winter holiday season. Using 16S ribosomal RNA gene amplicon sequencing, we demonstrated the feasibility of detecting sewage-derived human gut bacterial taxa involved in phytoestrogen metabolism, including Bifidobacterium, Blautia and Romboutsia. Combined metabolomic and genomic wastewater analysis can inform nutritional assessments at population scale, indicating wastewater-based epidemiology as a promising tool for actionable and cost-effective data collection to support public health nutrition.

Rhizobium Phage-Like Microvirus Genome Sequence Identified in Wastewater in Arizona, USA, in November 2020 Encodes an Endolysin and a Putative Multiheme Cytochrome c-like Protein.

We describe the genome (4,696 nucleotides [GC content, 56%; coverage, 3,641×) of MAZ-Nov-2020, a microvirus identified from municipal wastewater in Maricopa County, Arizona, USA, in November 2020. The MAZ-Nov-2020 genome encodes major capsid protein, endolysin, replication initiator protein, and two hypothetical proteins, one of which was predicted to likely be a membrane-associated multiheme cytochrome c.

Wastewater surveillance uncovers regional diversity and dynamics of SARS-CoV-2 variants across nine states in the USA.

Wastewater-based epidemiology (WBE) is a non-invasive and cost-effective approach for monitoring the spread of a pathogen within a community. WBE has been adopted as one of the methods to monitor the spread and population dynamics of the SARS-CoV-2 virus, but significant challenges remain in the bioinformatic analysis of WBE-derived data. Here, we have developed a new distance metric, CoVdist, and an associated analysis tool that facilitates the application of ordination analysis to WBE data and the identification of viral population changes based on nucleotide variants. We applied these new approaches to a large-scale dataset from 18 cities in nine states of the USA using wastewater collected from July 2021 to June 2022. We found that the trends in the shift between the Delta and Omicron SARS-CoV-2 lineages were largely consistent with what was seen in clinical data, but that wastewater analysis offered the added benefit of revealing significant differences in viral population dynamics at the state, city, and even neighborhood scales. We also were able to observe the early spread of variants of concern and the presence of recombinant lineages during the transitions between variants, both of which are challenging to analyze based on clinically-derived viral genomes. The methods outlined here will be beneficial for future applications of WBE to monitor SARS-CoV-2, particularly as clinical monitoring becomes less prevalent. Additionally, these approaches are generalizable, allowing them to be applied for the monitoring and analysis of future viral outbreaks.