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

Discrimination of Human and Non-Human Sources of Pollution in Gulf of Mexico Waters by Microbial Source Tracking Methods and the Investigation of the Influence of Environmental Factors on \u3ci\u3eEscherichia coli\u3c/i\u3e Survival

Water quality worldwide is assessed by enumeration of fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli, and enterococci) intended to act as surrogates for human enteric pathogens. In environmental waters, this predictive relationship is confounded by many possible sources of FIB with varying implications for human health. Many physico-chemical and biological factors influence the fate of enteric pathogens and FIB in aquatic habitats, but are poorly understood, thus limiting our understanding of the usefulness of FIB as fecal pollution indicators. These studies explored the field application of a “toolbox” approach to microbial source tracking (MST) intended to discriminate between human and non-human fecal pollution: a) in a Florida estuary used for shellfishing and recreational activities and b) at public beaches before and after remediation of wastewater infrastructure. Lastly, the effects of environmental factors (sediments, protozoa, sunlight) on survival of culturable E. coli were investigated in freshwater and seawater mesocosms simulating environmental conditions. Detection of a human- associated MST marker (the esp gene of Enterococcus faecium) at sites with suspected sewage contamination indicated that human fecal pollution is impacting water quality in Wakulla County, while Lagrangian drifters designed to follow current and tidal movement suggested that local hydrology plays an important role in bacterial transport and deposition pathways. Elevated FIB concentrations and frequent detection of human-associated MST markers (esp and human polyomaviruses) identified human sewage pollution at a public beach, facilitating remediation efforts (sewage main repair, removal of portable/abandoned restrooms), followed by significant decreases in FIB concentrations and MST marker detection. These studies show that comprehensive microbial water quality assessment can reliably identify contamination sources, thereby improving pollution mitigation and restoring recreational water quality. Protozoan predation, freshwater vs. seawater habitat and sediment vs. water column location affected the concentration of culturable E. coli in outdoor mesocosms. Sediments offered a refuge from predation where freshwater vs. seawater habitat was amore important determinant of survival. These findings provide important insight into the ecology of E. coli and their natural predators in aquatic habitats and underscore the inherent effect different habitats play in their survival

Relationships between Microbial Indicators and Pathogens in Recreational Water Settings

Fecal pollution of recreational waters can cause scenic blight and pose a threat to public health, resulting in beach advisories and closures. Fecal indicator bacteria (total and fecal coliforms, Escherichia coli, and enterococci), and alternative indicators of fecal pollution (Clostridium perfringens and bacteriophages) are routinely used in the assessment of sanitary quality of recreational waters. However, fecal indicator bacteria (FIB), and alternative indicators are found in the gastrointestinal tract of humans, and many other animals and therefore are considered general indicators of fecal pollution. As such, there is room for improvement in terms of their use for informing risk assessment and remediation strategies. Microbial source tracking (MST) genetic markers are closely associated with animal hosts and are used to identify fecal pollution sources. In this review, we examine 73 papers generated over 40 years that reported the relationship between at least one indicator and one pathogen group or species. Nearly half of the reports did not include statistical analysis, while the remainder were almost equally split between those that observed statistically significant relationships and those that did not. Statistical significance was reported less frequently in marine and brackish waters compared to freshwater, and the number of statistically significant relationships was considerably higher in freshwater (p \u3c 0.0001). Overall, significant relationships were more commonly reported between FIB and pathogenic bacteria or protozoa, compared to pathogenic viruses (p: 0.0022–0.0005), and this was more pronounced in freshwater compared to marine. Statistically significant relationships were typically noted following wet weather events and at sites known to be impacted by recent fecal pollution. Among the studies that reported frequency of detection, FIB were detected most consistently, followed by alternative indicators. MST markers and the three pathogen groups were detected least frequently. This trend was mirrored by reported concentrations for each group of organisms (FIB \u3e alternative indicators \u3e MST markers \u3e pathogens). Thus, while FIB, alternative indicators, and MST markers continue to be suitable indicators of fecal pollution, their relationship with waterborne pathogens, particularly viruses, is tenuous at best and influenced by many different factors such as frequency of detection, variable shedding rates, differential fate and transport characteristics, as well as a broad range of site-specific factors such as the potential for the presence of a complex mixture of multiple sources of fecal contamination and pathogens

A fully nonlinear viscohyperelastic model for the brain tissue applicable to dynamic rates

Understanding the mechanical response of the brain to external loadings is of critical importance in investigating the pathological conditions of this tissue during injurious conditions. Such injurious loadings may occur at high rates, for example among others, during road traffic or sport accidents, falls, or due to explosions. Hence, investigating the injury mechanism and design of protective devices for the brain requires constitutive modeling of this tissue at such rates. Accordingly, this paper is aimed at critically investigating the physical background for viscohyperelastic modeling of the brain tissue with scrutinizing the elastic fields pertinent to large, time dependent deformations, and developing a fully nonlinear multimode Maxwell model that can mathematically explain such deformations. The proposed model can be calibrated using the simple monotonic uniaxial deformation of the sample extracted from the tissue, and does not require additional information from relaxation or creep experiments. The performance of the proposed model is examined using the experimental results of two different studies, which reveals a desirable agreement. The usefulness, limitations, and future developments of the proposed model are discussed in this paper

An Indirect Indentation Method for Evaluating the Linear Viscoelastic Properties of the Brain Tissue

Indentation experiments offer a robust, fast, and repeatable testing method for evaluating the mechanical properties of the solid-state materials in a wide stiffness range. With the advantage of requiring a minimal sample preparation and multiple tests on a small piece of specimen, this method has recently become a popular technique for measuring the elastic properties of the biological materials, especially the brain tissue whose ultrasoft nature makes its mechanical characterization very challenging. Nevertheless, some limitations are associated with the indentation of the brain tissue, such as improper surface detection, negative initial contact force due to tip-tissue moisture interaction, and partial contact between the tip and the sample. In this study, an indirect indentation scheme is proposed to overcome the aforementioned difficulties. In this way, the indentation force is transferred from a sharp tip to the surface of the tissue slices via a rigid coverslip. To demonstrate the accuracy of this method, the linear viscoelastic properties of the white and gray matters of the bovine brain samples are measured by imposing small cyclic loads at different frequencies. The rate, regional, directional, and postmortem time dependence of the viscoelastic moduli are investigated and compared with the previous results from cyclic shear and monotonic experiments on the brain tissue. While findings of this research present a comprehensive set of information for the viscoelastic properties of the brain at a wide frequency range, the central goal of this paper is to introduce a novel experimentation technique with noticeable advantages for biomechanical characterization of the soft tissue

Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater

There is currently a clear benefit for many countries to utilize wastewater-based epidemiology (WBE) as part of ongoing measures to manage the coronavirus disease 2019 (COVID-19) global pandemic. Since most wastewater virus concentration methods were developed and validated for nonenveloped viruses, it is imperative to determine the efficiency of the most commonly used methods for the enveloped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Municipal wastewater seeded with a human coronavirus (CoV) surrogate, murine hepatitis virus (MHV), was used to test the efficiency of seven wastewater virus concentration methods: (A–C) adsorption-extraction with three different pre-treatment options, (D–E) centrifugal filter device methods with two different devices, (F) polyethylene glycol (PEG 8000) precipitation, and (G) ultracentrifugation. MHV was quantified by reverse-transcription quantitative polymerase chain reaction and the recovery efficiency was calculated for each method. The mean MHV recoveries ranged from 26.7 to 65.7%. The most efficient methods were adsorption-extraction methods with MgCl2 pre-treatment (Method C), and without pre-treatment (Method B). The third most efficient method used the Amicon® Ultra-15 centrifugal filter device (Method D) and its recovery efficiency was not statistically different from the most efficient methods. The methods with the worst recovery efficiency included the adsorption-extraction method with acidification (A), followed by PEG precipitation (F). Our results suggest that absorption-extraction methods with minimal or without pre-treatment can provide suitably rapid, cost-effective and relatively straightforward recovery of enveloped viruses in wastewater. The MHV is a promising process control for SARS-CoV-2 surveillance and can be used as a quality control measure to support community-level epidemic mitigation and risk assessment

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

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially 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 increasing rapidly. However, there are no standardized protocols or 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 cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR 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 when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases. Crown Copyright (C) 2021 Published by Elsevier B.V.Peer reviewe