Assessment of nucleic acid extraction protocols for antibiotic resistance genes (ARGs) quantification in aircraft wastewater.

This study evaluated ten nucleic acid extraction protocols (EP1 to EP10) for measuring five endogenous antibiotic resistance genes (ARGs) in four aircraft wastewater samples (AWW1 to AWW4). The targeted ARGs, including blaCTX-M, blaNDM-1, ermB, qnrS, and tetA, encompassed highly and minimally abundant ARGs. TetA and ermB were consistently detected across four aircraft wastewater samples using the DNeasy Blood and Tissue Kit and the AllPrep PowerViral DNA/RNA kit. QnrS displayed high detection rates with specific extraction protocols and aliquot volumes. Concentrations of ARGs varied across aircraft wastewater samples, with differing extraction protocols influencing quantitative results. The concentrations of tetA, ermB, and qnrS in AWW1 were distinct, while AWW2 to AWW4 exhibited a broader range for tetA, ermB, qnrS, blaCTX-M, and blaNDM-1. EP1 consistently produced the highest concentrations for several ARGs. Collective data analysis revealed varying ARG concentrations across the ten extraction protocols, suggesting the importance of careful extraction protocol selection in ARG monitoring in aircraft wastewater samples. Based on the results, we suggest that a small sample volume (as low as 0.2 mL) may be sufficient for ARG characterization in aircraft wastewater samples. The findings also emphasize the need for considering toilet paper removal without compromising nucleic acid extraction efficiency. The study highlights promising prospects for aircraft wastewater monitoring of ARGs, calling for further investigation into the import and spread of unique ARGs through transport hubs.

Research gaps and priorities for quantitative microbial risk assessment (QMRA).

The coronavirus disease 2019 pandemic highlighted the need for more rapid and routine application of modeling approaches such as quantitative microbial risk assessment (QMRA) for protecting public health. QMRA is a transdisciplinary science dedicated to understanding, predicting, and mitigating infectious disease risks. To better equip QMRA researchers to inform policy and public health management, an Advances in Research for QMRA workshop was held to synthesize a path forward for QMRA research. We summarize insights from 41 QMRA researchers and experts to clarify the role of QMRA in risk analysis by (1) identifying key research needs, (2) highlighting emerging applications of QMRA; and (3) describing data needs and key scientific efforts to improve the science of QMRA. Key identified research priorities included using molecular tools in QMRA, advancing dose-response methodology, addressing needed exposure assessments, harmonizing environmental monitoring for QMRA, unifying a divide between disease transmission and QMRA models, calibrating and/or validating QMRA models, modeling co-exposures and mixtures, and standardizing practices for incorporating variability and uncertainty throughout the source-to-outcome continuum. Cross-cutting needs identified were to: develop a community of research and practice, integrate QMRA with other scientific approaches, increase QMRA translation and impacts, build communication strategies, and encourage sustainable funding mechanisms. Ultimately, a vision for advancing the science of QMRA is outlined for informing national to global health assessments, controls, and policies.

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.

Highly socially vulnerable communities exhibit disproportionately increased viral loads as measured in community wastewater.

Wastewater surveillance has proven to be a useful tool for evidence-based epidemiology in the fight against the SARS-CoV-2 virus. It is particularly useful at the population level where acquisition of individual test samples may be time or cost-prohibitive. Wastewater surveillance for SARS-CoV-2 has typically been performed at wastewater treatment plants; however, this study was designed to sample on a local level to monitor the spread of the virus among three communities with distinct social vulnerability indices in Shreveport, Louisiana, located in a socially vulnerable region of the United States. Twice-monthly grab samples were collected from September 30, 2020, to March 23, 2021, during the Beta wave of the pandemic. The goals of the study were to examine whether: 1) concentrations of SARS-CoV-2 RNA in wastewater varied with social vulnerability indices and, 2) the time lag of spikes differed during wastewater monitoring in the distinct communities. The size of the population contributing to each sample was assessed via the quantification of the pepper mild mottle virus (PMMoV), which was significantly higher in the less socially vulnerable community. We found that the communities with higher social vulnerability exhibited greater viral loads as assessed by wastewater when normalized with PMMoV (Kruskal-Wallis, p < 0.05). The timing of the spread of the virus through the three communities appeared to be similar. These results suggest that interconnected communities within a municipality experienced the spread of the SARS-CoV-2 virus at similar times, but areas of high social vulnerability experienced more intense wastewater viral loads.

Producing ratio measures of effect with quantitative microbial risk assessment.

Estimating the risk of infections or other outcomes incident to pathogen exposure is a primary goal of quantitative microbial risk assessment (QMRA). Such estimates are useful to predict population-level risks, to evaluate exposures based on normative or tolerable risk guidelines, and to interpret the likely public health relevance of microbial measurements in environmental media. To evaluate alternative control measures (interventions), ratio estimates of effect (e.g., odds and risk ratios) are needed that are more broadly interpretable in the health sciences and consistent with convention in epidemiology. In this paper, we propose a general method for estimating widely used ratio measures of effect derived from stochastic QMRA approaches, including the generation of appropriate confidence intervals. Such QMRA-derived ratios can be used as a basis for evaluating interventions via hypothesis testing and for inclusion in systematic reviews and meta-analyses in a form consistent with risk estimation approaches commonly used in epidemiology.

Inferring SARS-CoV-2 RNA shedding into wastewater relative to the time of infection.

Since the start of the coronavirus disease-2019 (COVID-19) pandemic, there has been interest in using wastewater monitoring as an approach for disease surveillance. A significant uncertainty that would improve the interpretation of wastewater monitoring data is the intensity and timing with which individuals shed RNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into wastewater. By combining wastewater and case surveillance data sets from a university campus during a period of heightened surveillance, we inferred that individual shedding of RNA into wastewater peaks on average 6 days (50% uncertainty interval (UI): 6-7; 95% UI: 4-8) following infection, and that wastewater measurements are highly overdispersed [negative binomial dispersion parameter, k = 0.39 (95% credible interval: 0.32-0.48)]. This limits the utility of wastewater surveillance as a leading indicator of secular trends in SARS-CoV-2 transmission during an epidemic, and implies that it could be most useful as an early warning of rising transmission in areas where transmission is low or clinical testing is delayed or of limited capacity.

Quantitative Microbial Risk Assessment of Pediatric Infections Attributable to Ingestion of Fecally Contaminated Domestic Soils in Low-Income Urban Maputo, Mozambique.

Rigorous studies of water, sanitation, and hygiene interventions in low- and middle-income countries (LMICs) suggest that children are exposed to enteric pathogens via multiple interacting pathways, including soil ingestion. In 30 compounds (household clusters) in low-income urban Maputo, Mozambique, we cultured Escherichia coli and quantified gene targets from soils (E. coli: ybbW, Shigella/enteroinvasive E. coli (EIEC): ipaH, Giardia duodenalis: ß-giardin) using droplet digital PCR at three compound locations (latrine entrance, solid waste area, dishwashing area). We found that 88% of samples were positive for culturable E. coli (mean = 3.2 log10 CFUs per gram of dry soil), 100% for molecular E. coli (mean = 5.9 log10 gene copies per gram of dry soil), 44% for ipaH (mean = 2.5 log10), and 41% for ß-giardin (mean = 2.1 log10). Performing stochastic quantitative microbial risk assessment using soil ingestion parameters from an LMIC setting for children 12-23 months old, we estimated that the median annual infection risk by G. duodenalis was 7100-fold (71% annual infection risk) and by Shigella/EIEC was 4000-fold (40% annual infection risk) greater than the EPA's standard for drinking water. Compounds in Maputo, and similar settings, require contact and source control strategies to reduce the ingestion of contaminated soil and achieve acceptable levels of risk.

Detection and Quantification of Enteric Pathogens in Aerosols Near Open Wastewater Canals in Cities with Poor Sanitation.

Urban sanitation infrastructure is inadequate in many low-income countries, leading to the presence of highly concentrated, uncontained fecal waste streams in densely populated areas. Combined with mechanisms of aerosolization, airborne transport of enteric microbes and their genetic material is possible in such settings but remains poorly characterized. We detected and quantified enteric pathogen-associated gene targets in aerosol samples near open wastewater canals (OWCs) or impacted (receiving sewage or wastewater) surface waters and control sites in La Paz, Bolivia; Kanpur, India; and Atlanta, USA, via multiplex reverse-transcription qPCR (37 targets) and ddPCR (13 targets). We detected a wide range of enteric targets, some not previously reported in extramural urban aerosols, with more frequent detections of all enteric targets at higher densities in La Paz and Kanpur near OWCs. We report density estimates ranging up to 4.7 × 102 gc per mair3 across all targets including heat-stable enterotoxigenic Escherichia coli, Campylobacter jejuni, enteroinvasive E. coli/Shigella spp., Salmonella spp., norovirus, and Cryptosporidium spp. Estimated 25, 76, and 0% of samples containing positive pathogen detects were accompanied by culturable E. coli in La Paz, Kanpur, and Atlanta, respectively, suggesting potential for viability of enteric microbes at the point of sampling. Airborne transmission of enteric pathogens merits further investigation in cities with poor sanitation.

Antimicrobial resistance genes are enriched in aerosols near impacted urban surface waters in La Paz, Bolivia.

Antibiotic resistance poses a major global health threat. Understanding emergence and dissemination of antibiotic resistance in environmental media is critical to the design of control strategies. Because antibiotic resistance genes (ARGs) may be aerosolized from contaminated point sources and disseminated more widely in localized environments, we assessed ARGs in aerosols in urban La Paz, Bolivia, where wastewater flows in engineered surface water channels through the densely populated urban core. We quantified key ARGs and a mobile integron (MI) via ddPCR and E. coli spp. as a fecal indicator by culture over two years during both the rainy and dry seasons in sites near wastewater flows. ARG targets represented major antibiotic groups-tetracyclines (tetA), fluoroquinolines (qnrB), and beta-lactams (blaTEM)-and an MI (intI1) represented the potential for mobility of genetic material. Most air samples (82%) had detectable targets above the experimentally determined LOD: most commonly blaTEM and intI1 (68% and 47% respectively) followed by tetA and qnrB (17% and 11% respectively). ARG and MI densities in positive air samples ranged from 1.3 × 101 to 6.6 × 104 gene copies/m3 air. Additionally, we detected culturable E. coli in the air (52% of samples <1 km from impacted surface waters) with an average density of 11 CFU/m3 in positive samples. We observed decreasing density of blaTEM with increasing distance up to 150 m from impacted surface waters. To our knowledge this is the first study conducting absolute quantification and a spatial analysis of ARGs and MIs in ambient urban air of a city with contaminated surface waters. Environments in close proximity to urban wastewater flows in this setting may experience locally elevated concentrations of ARGs, a possible concern for the emergence and dissemination of antimicrobial resistance in cities with poor sanitation.

Intraday variability of indicator and pathogenic viruses in 1-h and 24-h composite wastewater samples: Implications for wastewater-based epidemiology.

We monitored the concentration of indicator viruses crAssphage and pepper mild mottle virus (PMMoV) and human pathogen adenovirus (HAdV) in influent from a wastewater treatment plant in Brisbane, Australia in 1-h and 24-h composite samples. Over three days of sampling, the mean concentration of crAssphage gene copies (GC)/mL in 24-h composite samples did not differ significantly (p = 0.72-0.92), while for PMMoV GC/mL (p value range: 0.0002-0.0321) and HAdV GC/mL (p value range: 0.0028-0.0068) significant differences in concentrations were observed on one day of sampling compared to the other two. For all three viruses, the variation observed in 1-h composite samples was greater than the variation observed in 24-h composite samples. For crAssphage, in 54.1% of 1-h composite samples, the concentration was less than that observed in 24-h composite samples; whereas for PMMoV and HAdV the concentration was less in 79.2 and 70.9% of 1-h composite samples, respectively, compared to the relevant 24-h composite samples. Similarly, the concentration of crAssphage in 1-h compared to 24-h composite samples did not differ (p = 0.1082) while the concentrations of PMMoV (p < 0.0001) and HAdV (p < 0.0001) in 1-h composite samples were significantly different from 24-h composite samples. These results suggest that 24-h composite samples offer increased analytical sensitivity and decreased variability compared to 1-h composite samples when monitoring wastewater, especially for pathogenic viruses with low infection rates within a community. Thus, for wastewater-based epidemiology applications, 24-h composite samples are less likely to produce false negative results and erroneous public health information.

Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates (k). The average T90 (time required for 1-log10 reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T90 for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58-43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.

Extended-spectrum beta-lactamase (ESBL)-positive Escherichia coli presence in urban aquatic environments in Kanpur, India.

In India, high rates of antibiotic consumption and poor sanitation infrastructure combine to pose a significant risk to the public through the environmental transmission of antimicrobial resistance (AMR). The WHO has declared extended-spectrum beta-lactamase (ESBL)-positive Escherichia coli a key indicator for the surveillance of AMR worldwide. In the current study, we measured the prevalence of AMR bacteria in an urban aquatic environment in India by detecting metabolically active ESBL-positive E. coli. Water samples were collected in duplicate from 16 representative environmental water sources including open canals, drains, and rivers around Kanpur, Uttar Pradesh. We detected culturable E. coli in environmental water at 11 (69%) of the sites. Out of the 11 sites that were positive for culturable E. coli, ESBL-producing E. coli was observed at 7 (64%). The prevalence of ESBL-producing E. coli detected in the urban aquatic environment suggests a threat of AMR bacteria to this region.

Intensive allochthonous inputs along the Ganges River and their effect on microbial community composition and dynamics.

Little is known about microbial communities in the Ganges River, India and how they respond to intensive anthropogenic inputs. Here we applied shotgun metagenomics sequencing to study microbial community dynamics and function in planktonic samples collected along an approximately 700 km river transect, including urban cities and rural settings in upstream waters, before and after the monsoon rainy season. Our results showed that 11%-32% of the microbes represented terrestrial, sewage and human inputs (allochthonous). Sewage inputs significantly contributed to the higher abundance, by 13-fold of human gut microbiome (HG) associated sequences and 2-fold of antibiotic resistance genes (ARGs) in the Ganges relative to other riverine ecosystems in Europe, North and South America. Metagenome-assembled genome sequences (MAGs) representing allochthonous populations were detectable and tractable across the river after 1-2 days of (downstream) transport (> 200 km apart). Only approximately 8% of these MAGs were abundant in U.S. freshwater ecosystems, revealing distinct biodiversity for the Ganges. Microbial communities in the rainy season exhibited increased alpha-diversity and spatial heterogeneity and showed significantly weaker distance-decay patterns compared with the dry season. These results advance our understanding of the Ganges microbial communities and how they respond to anthropogenic pollution.

Selecting Household Water Treatment Options on the Basis of World Health Organization Performance Testing Protocols.

The World Health Organization's International Scheme to Evaluate Household Water Treatment Technologies serves to benchmark microbiological performance of existing and novel technologies and processes for small-scale drinking water treatment according to a tiered system. There is widespread uncertainty around which tiers of performance are most appropriate for technology selection and recommendation in humanitarian response or for routine safe water programming. We used quantitative microbial risk assessment (QMRA) to evaluate attributable reductions in diarrheal disease burden associated with water treatment technologies meeting the three tiers of performance under this Scheme, across a range of conditions. According to mean estimates and under most modeling conditions, potential health gains attributable to microbiologically improved drinking water are realized at the middle tier of performance: "comprehensive protection: high pathogen removal (★★)" for each reference pathogen. The highest tier of performance may yield additional marginal health gains where untreated water is especially contaminated and where adherence is 100%. Our results highlight that health gains from improved efficacy of household water treatment technology remain marginal when adherence is less than 90%. While selection of water treatment technologies that meet minimum WHO efficacy recommendations for comprehensive protection against waterborne pathogens is critical, additional criteria for technology choice and recommendation should focus on potential for correct, consistent, and sustained use.

Estimating Infection Risks and the Global Burden of Diarrheal Disease Attributable to Intermittent Water Supply Using QMRA.

Intermittent water supply (IWS) is prevalent throughout low and middle-income countries. IWS is associated with increased microbial contamination and potentially elevated risk of waterborne illness. We used existing data sets to estimate the population exposed to IWS, assess the probability of infection using quantitative microbial risk assessment, and calculate the subsequent burden of diarrheal disease attributable to consuming fecally contaminated tap water from an IWS. We used reference pathogens Campylobacter, Cryptosporidium, and rotavirus as conservative risk proxies for infections via bacteria, protozoa, and viruses, respectively. Results indicate that the median daily risk of infection is an estimated 1 in 23 500 for Campylobacter, 1 in 5 050 000 for Cryptosporidium, and 1 in 118 000 for rotavirus. Based on these risks, IWS may account for 17.2 million infections causing 4.52 million cases of diarrhea, 109 000 diarrheal DALYs, and 1560 deaths each year. The burden of diarrheal disease associated with IWS likely exceeds the WHO health-based normative guideline for drinking water of 10-6 DALYs per person per year. Our results underscore the importance water safety management in water supplies and the potential benefits of point-of-use treatment to mitigate risks.

Leveraging wastewater surveillance to detect viral diseases in livestock settings.

Wastewater surveillance has evolved into a powerful tool for monitoring public health-relevant analytes. Recent applications in tracking severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection highlight its potential. Beyond humans, it can be extended to livestock settings where there is increasing demand for livestock products, posing risks of disease emergence. Wastewater surveillance may offer non-invasive, cost-effective means to detect potential outbreaks among animals. This approach aligns with the "One Health" paradigm, emphasizing the interconnectedness of animal, human, and ecosystem health. By monitoring viruses in livestock wastewater, early detection, prevention, and control strategies can be employed, safeguarding both animal and human health, economic stability, and international trade. This integrated "One Health" approach enhances collaboration and a comprehensive understanding of disease dynamics, supporting proactive measures in the Anthropocene era where animal and human diseases are on the rise.