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

Kinetics and Mechanisms of Human Adenovirus 2 Inactivation With UV and Chlorine

202 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007.In the United States, the EPA issues rules for water treatment plants, listing the pathogens that must be inactivated to ensure the safety of the water supply. A wide range of treatment strategies are used by the numerous water utilities because the most efficient methods are not standardized and because the byproducts of free chlorine are under increasing scrutiny. The methodology of water treatment is further complicated by emerging threats to potable water. Of particular interest are adenoviruses, which are on the CCL2 issued by the EPA. Unfortunately, the recently-adopted method of irradiation by ultraviolet (UV) light is largely ineffective against adenoviruses. Research is therefore required to find whether any of the other commonly-used treatments, separately or together, can be effective against this threat. I therefore tested the efficacy of monochloramine for inactivating adenoviruses at numerous variations of pH and temperature. Relatedly, a biological investigation of the mechanisms of virus inactivation (by UV light and monochloramine) was undertaken. Subsequently, a test of sequential treatment of virions, first by UV light and then by monochloramine, was performed to discover a possible synergistic effect. Finally, an alternative sequential treatment, first by decreased free chlorine and then by monochloramine, was also tested for efficacy. Biologically, I found that both monochloramine and UV irradiation inhibit an early phase of virus replication, a step prior to the synthesis of E1A. My extensive characterization of the effects of pH and temperature show that monochloramine accomplishes less inactivation of adenoviruses in conditions of high pH and/or low temperature. My tests also show that sequential treatments of either UV light/monochloramine or reduced dosage of free chlorine/monochloramine show no synergy in inactivating adenoviruses. As a result of this work, it is clear that novel disinfection strategies are required to meet the challenge of adenovirus disinfection.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Effect of Exposure to UV-C Irradiation and Monochloramine on Adenovirus Serotype 2 Early Protein Expression and DNA Replication▿

The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20°C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants

PCR data and comparative performance of Bacteroidales microbial source tracking genetic markers

We reported modified endpoint PCR results analyzed by universal and human-, swine-, and cattle-specific Bacteroidales gene markers with human sewage and animal fecal samples (i.e., swine, cattle, chicken, goat, sheep, buffalo, and duck) from Tha Chin and Chao Phraya watersheds. Annealing locations of PCR primers were illustrated by maps of 16s rRNA Bacteroidales genes. We also summarized previously published work on the performance of the PCR assays. For further discussion of the data presented here, please refer to Somnark et al., Performance evaluation of Bacteroidales genetic markers for human and animal microbial source tracking in tropical agricultural watersheds, Environ. Pollut. 236 (2018) 100–110. Keywords: Endpoint PCR, Fecal pollution, Microbial source tracking, Bacteroidales, Sensitivity, Specificity, Water qualit

Integrated Multivariate Analysis with Nondetects for the Development of Human Sewage Source-Tracking Tools Using Bacteriophages of <i>Enterococcus faecalis</i>

We developed sewage-specific microbial source tracking (MST) tools using enterococci bacteriophages and evaluated their performance with univariate and multivariate analyses involving data below detection limits. Newly isolated <i>Enterococci faecalis</i> bacterial strains AIM06 (DSM100702) and SR14 (DSM100701) demonstrated 100% specificity and 90% sensitivity to human sewage without detecting 68 animal manure pooled samples of cats, chickens, cows, dogs, ducks, pigs, and pigeons. AIM06 and SR14 bacteriophages were present in human sewage at 2–4 orders of magnitude. A principal component analysis confirmed the importance of both phages as main water quality parameters. The phages presented only in the polluted water, as classified by a cluster analysis, and at median concentrations of 1.71 × 10² and 4.27 × 10² PFU/100 mL, respectively, higher than nonhost specific RYC2056 phages and sewage-specific KS148 phages (<i>p</i> < 0.05). Interestingly, AIM06 and SR14 phages exhibited significant correlations with each other and with total coliforms, <i>E. coli</i>, enterococci, and biochemical oxygen demand (Kendall’s tau = 0.348 to 0.605, <i>p</i> < 0.05), a result supporting their roles as water quality indicators. This research demonstrates the multiregional applicability of enterococci hosts in MST application and highlights the significance of multivariate analysis with nondetects in evaluating the performance of new MST host strains

Monitoring and Modeling Non-Point Source Contributions of Host-Specific Fecal Contamination in San Pablo Bay

Fecal contamination from non-point sources in coastal and estuarine water bodies is a problem of increasing concern. Water monitoring alone is sometimes insufficient in providing a clear picture of the fecal contamination of a water body. Well-formulated and developed mathematical and numerical transport models, on the contrary, predict continuous concentrations of microbial indicators under diverse scenarios of interest, and they can quantify fecal source contributions on a land use basis (human versus livestock or wildlife).The present project has demonstrated the utility of quantitative analyses of fecal contamination in water bodies via new experimental measurement techniques as well as mathematical/numerical modeling of the fate and transport of biological contaminants. San Pablo Bay was selected because there is abundant knowledge about the estuarine system and its tributaries and because of the availability of an existing 3-dimensional model. Environmental monitoring of San Pablo Bay was performed with a Microbial Source Tracking (MST) approach that utilized a validated large-volume (100 L) hollowfiber ultrafiltration method to concentrate water, which was then tested with quantitative real-time PCR assays to identify and quantify fecal contamination contributed from general and human-, cow- and dog-specific sources. Monitoring of human viral pathogens (adeno- and enteroviruses) was also performed. Additional samples were also collected to enumerate microbial indicators (E. coli and enterococci). This was the first time that DNA-based molecular assays, which had been previously validated with fecal samples and wastewater effluents in freshwater studies, were applied to an estuarine environment.Monitoring results indicated low-level general and human-derived fecal contamination in the bay, while cow- and dog-derived contamination was not detected, except for one sample which contained dog-specific genetic marker. Human viruses were also below the sample detection limit. The pollution was more likely to come from surrounding urban areas or wastewater treatment facilities than from agricultural farm land or wildlife areas. Another application of the study included the validation of quantitative sea bird-specific molecular DNA assays for the purpose of microbial source tracking to enumerate the contribution of sea birds to fecal contamination. The assay was found to possess adequate sensitivity and specificity and could be applied in the future to archived environmental samples to estimate the contribution from sea birds to bacterial fecal loads in San Pablo Bay and its tributaries.A suite of theoretical and computational tools, ranging from one-dimensional (1-D) to three-dimensional (3-D) models, was developed during this project for the analysis of the fate and transport of Bacteroidales gene markers. These models were paired with existing visualization tools. Flow conditions for the sampling dates were simulated and results compared with velocity observations. Exploratory assessments of water quality in the San Pablo Bay allowed for an incipient characterization of the transport conditions in San Pablo Bay. Future work includes the optimization of the water-quality sub-module of the code in order to a) include sediment transport in the bay; b) obtain more reliable results 3 under a wide set of boundary conditions; and c) be able to simulate a wider set of waterquality parameters. Also, the authors would like to add a user-friendly interface to the code to facilitate decision making by coastal managers

Simultaneous Detection of Feces-specific Bacteriophages of Bacteroides fragilis with a Duplex PCR Assay

Bacteriophages of the Bacteroides fragilis strains HSP40 and RYC2056 are used as indicators of human-specific and general (non-host specific) fecal pollution in water bodies. However, conventional anaerobic cultivation methods require 1-2 days of incubation. To overcome this limitation, in this study, we developed a DNA-based method to simultaneously detect representative bacteriophages (B40-8 and B56-3) that infect B. fragilis strains HSP40 and RYC2056, respectively. Both phages yielded a 224-bp amplicon with the primer pair BT5414/BT5415, and an additional 152-bp PCR product was observed for B40-8 with the primer pair BT5579/BT5580. The detection limits for B40-8 and B56-3 were 10-5 and 10-4 ng of pure DNA, and 1 and 50 ng of DNA template when 5 and 5,000 PFU/mL were spiked into distilled water, respectively. The assay exhibited a higher sensitivity for sewage samples, with < 0.1 and 15 PFU/mL of phages infecting HSP40 and RYC2056, respectively. The assay did not produce false positive results for the Bacteroides phages PG76, HB13, and GA17 or for the enterococcal phages AIM06 and SR14. The assay also detected RYC2056 phages that were isolated from sewage samples and the phage B40-8 when it was spiked into raw sewage. Thus, the newly developed PCR assay demonstrated potential for the environmental monitoring of Bacteroides bacteriophages, decreasing the analysis time to a few hours

Outbreaks of Tilapia Lake Virus Infection, Thailand, 2015–2016

During 2015–2016, several outbreaks of tilapia lake virus infection occurred among tilapia in Thailand. Phylogenetic analysis showed that the virus from Thailand grouped with a tilapia virus (family Orthomyxoviridae) from Israel. This emerging virus is a threat to tilapia aquaculture in Asia and worldwide