The Effects of Sample Storage Conditions on the Microbial Community Composition in Hydraulic Fracturing Produced Water

The petroleum industry has an increasing interest in understanding the microbial communities driving biofouling and biocorrosion in reservoirs, wells, and infrastructure. However, sampling of the relevant produced fluids from subsurface environments for microbiological analyses is often challenged by high liquid pressures, workplace regulations, operator liability concerns, and remote sampling locations. These challenges result in infrequent sampling opportunities and the need to store and preserve the collected samples for several days or weeks. Maintaining a representative microbial community structure from produced fluid samples throughout storage and handling is essential for accurate results of downstream microbial analyses. Currently, no sample handling or storage recommendations exist for microbiological analyses of produced fluid samples. We used 16S rRNA gene sequencing to monitor the changes in microbial communities in hypersaline produced water stored at room temperature or at 4 oC for up to 7 days. We also analyzed storage at -80 oC across a 3-week period. The results suggest ideal handling methods would include placing the collected sample on ice as soon as possible, but at least within 24 h, followed by shipping the samples on ice over 2–3 days, and finally, long-term storage in the -20 oC or -80 oC freezer

Disinfection of Ebola Virus in Sterilized Municipal Wastewater

Concerns have been raised regarding handling of Ebola virus contaminated wastewater, as well as the adequacy of proposed disinfection approaches. In the current study, we investigate the inactivation of Ebola virus in sterilized domestic wastewater utilizing sodium hypochlorite addition and pH adjustment. No viral inactivation was observed in the one-hour tests without sodium hypochlorite addition or pH adjustment. No virus was recovered after 20 seconds (i.e. 4.2 log10 unit inactivation to detection limit) following the addition of 5 and 10 mg L-1 sodium hypochlorite, which resulted in immediate free chlorine residuals of 0.52 and 1.11 mg L-1, respectively. The addition of 1 mg L-1 sodium hypochlorite resulted in an immediate free chlorine residual of 0.16 mg L-1, which inactivated 3.5 log10 units of Ebola virus in 20 seconds. Further inactivation was not evident due to the rapid consumption of the chlorine residual. Elevating the pH to 11.2 was found to significantly increase viral decay over ambient conditions. These results indicate the high susceptibility of the enveloped Ebola virus to disinfection in the presence of free chlorine in municipal wastewater; however, we caution that extension to more complex matrices (e.g. bodily fluids) will require additional verification

Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance

Due to the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to generate large datasets aimed at detecting and quantifying SARS-CoV-2 RNA in wastewater. Although RT-qPCR is rapid and sensitive, there is no standard method yet, there are no certified quantification standards, and experiments are conducted using different assays, reagents, instruments, and data analysis protocols. These variations can induce errors in quantitative data reports, thereby potentially misleading interpretations, and conclusions. We review the SARS-CoV-2 wastewater surveillance literature focusing on variability of RT-qPCR data as revealed by inconsistent standard curves and associated parameters. We find that variation in these parameters and deviations from best practices, as described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines suggest a frequent lack of reproducibility and reliability in quantitative measurements of SARS-CoV-2 RNA in wastewater

Intestinal Interleukin-17 Receptor Signaling Mediates Reciprocal Control of the Gut Microbiota and Autoimmune Inflammation

Interleukin-17 (IL-17) and IL-17 receptor (IL-17R) signaling are essential for regulating mucosal host defense against many invading pathogens. Commensal bacteria, especially segmented filamentous bacteria (SFB), are a crucial factor that drives T helper 17 (Th17) cell development in the gastrointestinal tract. In this study, we demonstrate that Th17 cells controlled SFB burden. Disruption of IL-17R signaling in the enteric epithelium resulted in SFB dysbiosis due to reduced expression of α-defensins, Pigr and Nox1. When subjected to experimental autoimmune encephalomyelitis, IL-17R signaling deficient mice demonstrated earlier disease onset and worsened severity that was associated with increased intestinal Csf2 expression and elevated systemic GM-CSF cytokine concentrations. Conditional deletion of IL-17R in the enteric epithelium demonstrated that there was a reciprocal relationship between the gut microbiota and enteric IL-17R signaling that controlled dysbiosis, constrained Th17 development, and regulated the susceptibility to autoimmune inflammation

Monkeypox Outbreak: Wastewater and Environmental Surveillance Perspective

Monkeypox disease (MPXD), a viral disease caused by monkeypox virus (MPXV), is an emerging zoonotic disease endemic in some countries of Central and Western Africa but seldom reported outside the affected region. Since May 2022, MPXD has been reported at least in 74 countries globally, prompting the World Health Organization to declare the MPXD outbreak a Public Health Emergency of International Concern. As of July 24, 2022, 92% (68/74) of the countries with reported MPXD cases had no historical MPXD case reports. From the One Health perspective, the spread of MPXV in the environment poses a risk not only to humans but also to small mammals and may, ultimately, spread to potent novel host populations. Wastewater-based surveillance (WBS), has been extensively utilized for monitoring communicable diseases, particularly during the ongoing coronavirus disease, the COVID-19 pandemic It helped to monitor infectious disease caseloads as well as specific viral variants circulating in communities. The detection of MPXV DNA in various body fluids, including respiratory and nasal secretions, saliva, urine, feces, and semen of infected individuals, supports the possibility of using WBS as an early proxy for the detection of MPXV infections. WBS of MPXV DNA can be used to monitor MPXV activity/trends in sewerage network areas even before detecting laboratory-confirmed clinical cases within a community. However, several factors affect the detection of MPXV in wastewater including, but not limited to, routes and duration time of virus shedding by infected individuals, infection rates in the relevant affected population, environmental persistence, the processes and analytical sensitivity of the used methods. Further research is needed to identify the key factors that impact the detection of MPXV biomarkers in wastewater and improve the utility of WBS of MPXV as an early warning and monitoring tool for safeguarding human health. In this review, we shortly summarize aspects of MPXV outbreak relevant to wastewater monitoring and discuss the challenges associated with WBS.Peer reviewe

Monkeypox outbreak : Wastewater and environmental surveillance perspective

Monkeypox disease (MPXD), a viral disease caused by the monkeypox virus (MPXV), is an emerging zoonotic disease endemic in some countries of Central and Western Africa but seldom reported outside the affected region. Since May 2022, MPXD has been reported at least in 74 countries globally, prompting the World Health Organization to declare the MPXD outbreak a Public Health Emergency of International Concern. As of July 24, 2022; 92 % (68/74) of the countries with reported MPXD cases had no historical MPXD case reports. From the One Health perspective, the spread of MPXV in the environment poses a risk not only to humans but also to small mammals and may, ultimately, spread to potent novel host populations. Wastewater-based surveillance (WBS) has been extensively utilized to monitor communicable diseases, particularly during the ongoing COVID-19 pandemic. It helped in monitoring infectious disease caseloads as well as specific viral variants circulating in communities. The detection of MPXV DNA in lesion materials (e.g. skin, vesicle fluid, crusts), skin rashes, and various body fluids, including respiratory and nasal secretions, saliva, urine, feces, and semen of infected individuals, supports the possibility of using WBS as an early proxy for the detection of MPXV infections. WBS of MPXV DNA can be used to monitor MPXV activity/trends in sewerage network areas even before detecting laboratory-confirmed clinical cases within a community. However, several factors affect the detection of MPXV in wastewater including, but not limited to, routes and duration time of virus shedding by infected individuals, infection rates in the relevant affected population, environmental persistence, the processes and analytical sensitivity of the used methods. Further research is needed to identify the key factors that impact the detection of MPXV biomarkers in wastewater and improve the utility of WBS of MPXV as an early warning and monitoring tool for safeguarding human health. In this review, we shortly summarize aspects of the MPXV outbreak relevant to wastewater monitoring and discuss the challenges associated with WBS.publishedVersionPeer reviewe

Human Occupancy as a Source of Indoor Airborne Bacteria

Exposure to specific airborne bacteria indoors is linked to infectious and noninfectious adverse health outcomes. However, the sources and origins of bacteria suspended in indoor air are not well understood. This study presents evidence for elevated concentrations of indoor airborne bacteria due to human occupancy, and investigates the sources of these bacteria. Samples were collected in a university classroom while occupied and when vacant. The total particle mass concentration, bacterial genome concentration, and bacterial phylogenetic populations were characterized in indoor, outdoor, and ventilation duct supply air, as well as in the dust of ventilation system filters and in floor dust. Occupancy increased the total aerosol mass and bacterial genome concentration in indoor air PM10 and PM2.5 size fractions, with an increase of nearly two orders of magnitude in airborne bacterial genome concentration in PM10. On a per mass basis, floor dust was enriched in bacterial genomes compared to airborne particles. Quantitative comparisons between bacterial populations in indoor air and potential sources suggest that resuspended floor dust is an important contributor to bacterial aerosol populations during occupancy. Experiments that controlled for resuspension from the floor implies that direct human shedding may also significantly impact the concentration of indoor airborne particles. The high content of bacteria specific to the skin, nostrils, and hair of humans found in indoor air and in floor dust indicates that floors are an important reservoir of human-associated bacteria, and that the direct particle shedding of desquamated skin cells and their subsequent resuspension strongly influenced the airborne bacteria population structure in this human-occupied environment. Inhalation exposure to microbes shed by other current or previous human occupants may occur in communal indoor environments

Standardizing data reporting in the research community to enhance the utility of open data for SARS-CoV-2 wastewater surveillance

SARS-CoV-2 RNA detection in wastewater is being rapidly developed and adopted as a public health monitoring tool worldwide. With wastewater surveillance programs being implemented across many different scales and by many different stakeholders, it is critical that data collected and shared are accompanied by an appropriate minimal amount of meta-information to enable meaningful interpretation and use of this new information source and intercomparison across datasets. While some databases are being developed for specific surveillance programs locally, regionally, nationally, and internationally, common globally-adopted data standards have not yet been established within the research community. Establishing such standards will require national and international consensus on what meta-information should accompany SARS-CoV-2 wastewater measurements. To establish a recommendation on minimum information to accompany reporting of SARS-CoV-2 occurrence in wastewater for the research community, the United States National Science Foundation (NSF) Research Coordination Network on Wastewater Surveillance for SARS-CoV-2 hosted a workshop in February 2021 with participants from academia, government agencies, private companies, wastewater utilities, public health laboratories, and research institutes. This report presents the primary two outcomes of the workshop: (i) a recommendation on the set of minimum meta-information that is needed to confidently interpret wastewater SARS-CoV-2 data, and (ii) insights from workshop discussions on how to improve standardization of data reporting

Wastewater-based epidemiological surveillance to monitor the prevalence of SARS-CoV-2 in developing countries with onsite sanitation facilities

Wastewater-based epidemiology (WBE) has emerged as a valuable approach for forecasting disease outbreaks in developed countries with a centralized sewage infrastructure. On the other hand, due to the absence of well-defined and systematic sewage networks, WBE is challenging to implement in developing countries like Bangladesh where most people live in rural areas. Identification of appropriate locations for rural Hotspot Based Sampling (HBS) and urban Drain Based Sampling (DBS) are critical to enable WBE based monitoring system. We investigated the best sampling locations from both urban and rural areas in Bangladesh after evaluating the sanitation infrastructure for forecasting COVID-19 prevalence. A total of 168 wastewater samples were collected from 14 districts of Bangladesh during each of the two peak pandemic seasons. RT-qPCR commercial kits were used to target ORF1ab and N genes. The presence of SARS-CoV-2 genetic materials was found in 98% (165/168) and 95% (160/168) wastewater samples in the first and second round sampling, respectively. Although wastewater effluents from both the marketplace and isolation center drains were found with the highest amount of genetic materials according to the mixed model, quantifiable SARS-CoV-2 RNAs were also identified in the other four sampling sites. Hence, wastewater samples of the marketplace in rural areas and isolation centers in urban areas can be considered the appropriate sampling sites to detect contagion hotspots. This is the first complete study to detect SARS-CoV-2 genetic components in wastewater samples collected from rural and urban areas for monitoring the COVID-19 pandemic. The results based on the study revealed a correlation between viral copy numbers in wastewater samples and SARS-CoV-2 positive cases reported by the Directorate General of Health Services (DGHS) as part of the national surveillance program for COVID-19 prevention. The findings of this study will help in setting strategies and guidelines for the selection of appropriate sampling sites, which will facilitate in development of comprehensive wastewater-based epidemiological systems for surveillance of rural and urban areas of low-income countries with inadequate sewage infrastructure.This research was supported by Water Aid Bangladesh, North South University, Dhaka, COVID-19 Diagnostic Lab, Department of Microbiology, Noakhali Science and Technology University (NSTU), Noakhali, Bangladesh, the International Training Network of Bangladesh University of Engineering and Technology (ITN-BUET) - Centre for Water Supply and Waste Management, and KTH Royal Institute of Technology, Sweden. We acknowledge the sincere help and support of the staff and volunteers of NSTU-COVID-19 Diagnostic Lab, Noakhali Science and Technology University, Bangladesh during the different phases of the study. PB and MTI acknowledge the Life Science Technology Platform, Science for Life Laboratory for the seed funding to initiate the wastewater-based epidemiological studies for SARS-CoV-2 in Bangladesh. We would also like to acknowledge the two anonymous reviewers for their critical comments as well as their thoughtful insights, which has significantly improved the manuscript.Peer reviewe

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

Wastewater surveillance for pathogens using the reverse transcription-polymerase chain reaction (RT-PCR) is an effective, resource-efficient tool for gathering additional community-level public health information, including the incidence and/or prevalence and trends of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater may provide an early-warning signal of COVID-19 infections in a community. The capacity of the world’s environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is rapidly increasing. However, there are no standardized protocols nor harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can lead to false-positive and -negative errors in the surveillance of SARS-CoV-2, culminating in recommendations and strategies that can be implemented to identify and mitigate these errors. Recommendations include, stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly during a low incidence of SARS-CoV-2 in wastewater. Corrective and confirmatory actions must be in place for inconclusive and/or potentially significant results (e.g., initial onset or reemergence of COVID-19 in a community). It will also be prudent to perform inter-laboratory comparisons to ensure results are reliable and interpretable for ongoing and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance was demonstrated during this global crisis. In the future, wastewater will play an important role in the surveillance of a range of other communicable diseases.Highlights: Harmonized QA/QC procedures for SARS-CoV-2 wastewater surveillance are lacking; Wastewater analysis protocols are not optimized for trace analysis of viruses; False-positive and -negative errors have consequences for public health responses; Inter-laboratory studies utilizing standardized reference materials and protocols are needed.info:eu-repo/semantics/publishedVersio