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 fecal sources with rapid methods in coastal waters and sediments

Microbial contamination in coastal waters is an important public health and economic problem worldwide. It is common for the sources as well as the environmental fate and transport of fecal contamination in recreational waters to be unknown making it difficult to mitigate the input and to understand the relative health risk associated with a given water body. A comprehensive understanding of sources and bacterial dynamics is needed for effective mitigation and management of microbial contaminants.The main objectives of this study were to identify sources of fecal contamination to a chronically impaired Southern California watershed, to demonstrate applicability of microbial source tracking (MST) tools, including source-specific markers for discrimination of human and non-human sources, to evaluate the fate of fecal contaminates in coastal sediments, and to develop and optimize immunomagnetic separation/adenosine triphosphate (IMS/ATP) assays for rapid enumeration of viable fecal contamination. A three-year MST study was conducted to help explain elevated levels of surfzone fecal indicator bacteria (FIB) at Topanga State Beach, a critically impaired Southern California Beach. This study investigated sources of FIB to the Topanga watershed and the applicability of using MST technology longer time scales. MST markers effectively elucidated temporal and seasonal trends in fecal bacterial levels, and dog and gull marker appeared to be a significant sources to Topanga lagoon and Topanga State Beach. However, a lack of correlation between FIB and marker measurements was noted, and dog marker and FIB levels did not covary when compared at different Southern California beaches. Sediments were found to play an important and variable role in environmental fate of MST markers and FIB. Variable decay was observed for different indicators and in different sediments, with differences noted even within one watershed. The human HF183 marker was useful for providing evidence of recent inputs of human fecal contamination and behaved similarly to the molecular marker for Campylobacter (qCAMP) and FIB in brackish sediments. The general Bacteroides (GB3) and enterococci (ENT1A) markers were more conservative and under certain circumstances had comparable decay to culturable FIB. Application of a suite of markers may be necessary for effective evaluation of sediment fecal bacterial levels. Moreover, differences were observed between relative decay amongst the different sediments tested, illustrating the need for more routine sediment monitoring.IMS/ATP assays provided useful information regarding fecal contamination levels and measurements made had a consistent relationship with measurements made by standard methods. IMS/ATP utilizes paramagnetic beds and target-specific antibodies to isolate target organisms. Following isolation, adenosine tri-phosphate (ATP) is extracted from the target population and quantified. The Cov-IMS/ATP method rapidly measured viable enterococci in complex surface waters, providing a useful field tool for assessment of coastal water quality and for identification of hot spots of fecal contamination. An inversely-coupled (Inv-IMS/ATP) assay for detection of Bacteroides thetaiotaomicron was developed and applied for rapid detection of human-associated fecal contamination. The Inv-IMS/ATP assay yielded measurements of viable B. thetaiotaomicron that were comparable to the HF183 human marker in complex source waters impacted with both wastewater and runoff, and the Inv-IMS/ATP assay was able to effectively differentiate between surface waters impacted with adequately and inadequately treated wastewater. IMS/ATP assays show promise for rapid evaluation of recreational water quality in areas where access to more expensive methods is limited and in areas where water quality is unpredictable. This research highlights the difficulties and complexities associated with effective tracking and management of microbial contaminates in the coastal environment. Additional research evaluating relative aging of molecular markers and relative contributions from different sources is needed to fully interpret field-based source marker data. Sediments were shown to have an important and variable role in fate of fecal contaminants in the environment. Additional studies are needed evaluating how watershed models can most effectively be adapted to include a sediment compartment and how different sources of fecal contamination and source markers decay in sediments with variable characteristics. IMS/ATP assays showed promise and can be successfully applied in complex waters for rapid enumeration of viable fecal contamination; additional verification of assay performance is needed at complex sites impacted with multiple sources

Evaluation of approaches to quantify total residual oxidants in ballast water management systems employing chlorine for disinfection.

With the maturation and certification of several ballast water management systems that employ chlorine as biocide to prevent the spread of invasive species, there is a clear need for accurate and reliable total residual oxidants (TRO) technology to monitor treatment dose and assure the environmental safety of treated water discharged from ships. In this study, instruments used to measure TRO in wastewater and drinking water applications were evaluated for their performance in scenarios mimicking a ballast water treatment application (e.g., diverse hold times, temperatures, and salinities). Parameters chosen for testing these technologies in the past do not reflect conditions expected during ballast water treatment. Salinity, temperature, and oxidant concentration all influenced the response of amperometric sensors. Oxidation reduction potential (ORP) sensors performed more consistently than amperometric sensors under different conditions but it may be difficult to correlate ORP and TRO measurements for the multitude of biogeochemical conditions found naturally in ballast water. N,N-diethyl-p-phenylenediamine (DPD) analyzers and amperometric sensors were also tested under intermittent sampling conditions mimicking a ballasting scenario, with cyclical dosage and discharge operations. When sampling was intermittent, amperometric sensors required excessive response and conditioning times, whereas DPD analyzers provided reasonable estimates of TRO under the ballasting scenario

Dry and Wet Weather Survey for Human Fecal Sources in the San Diego River Watershed

State and federal agencies regulate fecal indicator bacteria (FIB), such as E. coli or Enterococcus, in order to manage public health risks at swimming beaches. Despite these goals, watershed managers are challenged in terms of how to best clean up sources of FIB because concentrations frequently exceed water quality objectives, and sources—both human and nonhuman sources of FIB—appear to be everywhere. Since most nonhuman fecal sources represent substantially lower public health risks than human sources do, this study utilizes the human fecal source marker HF183 to better define watershed managers’ riskiest sites and times in order to prioritize remediation actions. A total of 117 samples were collected and analyzed for both FIB and HF183 from 26 sites during multiple sampling campaigns between 2019 and 2021 along the mainstem in addition to major tributaries in a highly urbanized watershed. The results indicated that the vast majority of samples (96%) quantified HF183 during wet weather, ranging from 99 to 44,768 gene copies/100 mL. Similar to HF183, the FIB results exceeded water quality objectives for 100% of the samples in wet weather; however, HF183 was rarely quantified in dry weather, with 3 of 72 samples (4%) exceeding 500 gene copies/100 mL, while two-thirds of samples (67%) exceeded FIB water quality objectives during dry weather. Where HF183 was detected in dry weather, isolated and unpredictable events explained human fecal pollution. It is more challenging in wet weather to identify and quantify the source(s) of human fecal pollution

Performance of a One-Dimensional Model of Wave-Driven Nearshore Alongshore Tracer Transport and Decay with Applications for Dry Weather Coastal Pollution

Dry weather pollution sources cause coastal water quality problems that are not accounted for in existing beach advisory metrics. A 1D wave-driven advection and loss model was developed for a 30 km nearshore domain spanning the United States/Mexico border region. Bathymetric nonuniformities, such as the inlet and shoal near the Tijuana River estuary mouth, were neglected. Nearshore alongshore velocities were estimated by using wave properties at an offshore location. The 1D model was evaluated using the hourly output of a 3D regional hydrodynamic model. The 1D model had high skill in reproducing the spatially averaged alongshore velocities from the 3D model. The 1D and 3D models agreed on tracer exceedance or nonexceedance above a human illness probability threshold for 87% of model time steps. 1D model tracer was well-correlated with targeted water samples tested for DNA-based human fecal indicators. This demonstrates that a simple, computationally fast, 1D nearshore wave-driven advection model can reproduce nearshore tracer evolution from a 3D model over a range of wave conditions ignoring bathymetric nonuniformities at this site and may be applicable to other locations

Large-scale implementation of standardized quantitative real-time PCR fecal source identification procedures in the Tillamook Bay Watershed.

Fecal pollution management remains one of the biggest challenges for water quality authorities worldwide. Advanced fecal pollution source identification technologies are now available that can provide quantitative information from many animal groups. As public interest in these methodologies grows, it is vital to use standardized procedures with clearly defined data acceptance metrics and conduct field studies demonstrating the use of these techniques to help resolve real-world water quality challenges. Here we apply recently standardized human-associated qPCR methods with custom data acceptance metrics (HF183/BacR287 and HumM2), along with established procedures for ruminant (Rum2Bac), cattle (CowM2 and CowM3), canine (DG3 and DG37), and avian (GFD) fecal pollution sources to (i) demonstrate the feasibility of implementing standardized qPCR procedures in a large-scale field study, and (ii) characterize trends in fecal pollution sources in the research area. A total of 602 water samples were collected over a one-year period at 29 sites along the Trask, Kilchis, and Tillamook rivers and tributaries in the Tillamook Bay Watershed (OR, USA). Host-associated qPCR results were combined with high-resolution geographic information system (GIS) land use and general indicator bacteria (E. coli) measurements to elucidate water quality fecal pollution trends. Results demonstrate the feasibility of implementing standardized fecal source identification qPCR methods with established data acceptance metrics in a large-scale field study leading to new investigative leads suggesting that elevated E. coli levels may be linked to specific pollution sources and land use activities in the Tillamook Bay Watershed

RNA Viromics of Southern California Wastewater and Detection of SARS-CoV-2 Single-Nucleotide Variants.

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the coronavirus disease 2019 (COVID-19) pandemic to track disease burden through analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the health care system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven wastewater treatment plants (WTPs), collected from August 2020 to January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single-nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater, and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically important SARS-CoV-2 variants along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. IMPORTANCE Wastewater-based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the coronavirus disease 2019 (COVID-19) pandemic to track the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) without adding burden to health care systems. In this study, we used metatranscriptomics and reverse transcription-droplet digital PCR (RT-ddPCR) to assay RNA viruses across Southern California wastewater from August 2020 to January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single-nucleotide variants through sequencing. Likewise, wastewater treatment plants (WTPs) harbored different viromes, and we detected other human pathogens, such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology

Longitudinal metatranscriptomic sequencing of Southern California wastewater representing 16 million people from August 2020-21 reveals widespread transcription of antibiotic resistance genes

Municipal wastewater provides a representative sample of human fecal waste across a catchment area and contains a wide diversity of microbes. Sequencing wastewater samples provides information about human-associated and medically-important microbial populations, and may be useful to assay disease prevalence and antimicrobial resistance (AMR). Here, we present a study in which we used untargeted metatranscriptomic sequencing on RNA extracted from 275 sewage influent samples obtained from eight wastewater treatment plants (WTPs) representing approximately 16 million people in Southern California between August 2020 – August 2021. We characterized bacterial and viral transcripts, assessed metabolic pathway activity, and identified over 2,000 AMR genes/variants across all samples. Because we did not deplete ribosomal RNA, we have a unique window into AMR carried as ribosomal mutants. We show that AMR diversity varied between WTPs and that the relative abundance of many individual AMR genes/variants increased over time and may be connected to antibiotic use during the COVID-19 pandemic. Similarly, we detected transcripts mapping to human pathogenic bacteria and viruses suggesting RNA sequencing is a powerful tool for wastewater-based epidemiology and that there are geographical signatures to microbial transcription. We captured the transcription of gene pathways common to bacterial cell processes, including central carbon metabolism, nucleotide synthesis/salvage, and amino acid biosynthesis. We also posit that due to the ubiquity of many viruses and bacteria in wastewater, new biological targets for microbial water quality assessment can be developed. To the best of our knowledge, our study provides the most complete longitudinal metatranscriptomic analysis of a large population’s wastewater to date and demonstrates our ability to monitor the presence and activity of microbes in complex samples. By sequencing RNA, we can track the relative abundance of expressed AMR genes/variants and metabolic pathways, increasing our understanding of AMR activity across large human populations and sewer sheds