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.

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.

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.

Comparison of concentration and extraction workflows for qPCR quantification of intI1 and vanA in untreated wastewater.

Quantitative polymerase chain reaction (qPCR) measurement of antibiotic resistance genes (ARGs) in untreated municipal wastewater may prove useful in combating the antimicrobial resistance crisis. However, harmonizing and optimizing qPCR-based workflows is essential to facilitate comparisons across studies, and includes achieving highly-effective ARG capture through efficient concentration and extraction procedures. In the current study, combinations of sample volume, membrane types and DNA extraction kits within filtration and centrifugation-based workflows were used to quantify 16S ribosomal RNA (16S rRNA), class 1 integron-integrase gene (intI1) and an ARG encoding resistance to vancomycin (vanA) in untreated wastewater sampled from three wastewater treatment plants (WWTPs). Highly abundant 16S rRNA and intI1 were detected in 100 % of samples from all three WWTPs using both 2 and 20 mL sample volumes, while lower prevalence vanA was only detected when using the 20 mL volume. When filtering 2 mL of wastewater, workflows with 0.20-/0.40-µm polycarbonate (PC) membranes generally yielded greater concentrations of the three targets than workflows with 0.22-/0.45-µm mixed cellulose ester (MCE) membranes. The improved performance was diminished when the sample volume was increased to 20 mL. Consistently greater concentrations of 16S rRNA, intI1 and vanA were yielded by filtration-based workflows using PC membranes combined with a DNeasy PowerWater (DPW) Kit, regardless of the sample volume used, and centrifugation-based workflows with DNeasy Blood & Tissue Kit for 2-mL wastewater extractions. Within the filtration-based workflows, the DPW kit yielded more detection and quantifiable results for less abundant vanA than the DNeasy PowerSoil Pro Kit and FastDNA™ SPIN Kit for Soil. These findings indicate that the performance of qPCR-based workflows for surveillance of ARGs in wastewater varies across targets, sample volumes, concentration methods and extraction kits. Workflows must be carefully considered and validated considering the target ARGs to be monitored.

Environmental DNA (eDNA) removal rates in streams differ by particle size under varying substrate and light conditions.

The use of environmental DNA (eDNA) as a sampling tool offers insights into the detection of invasive and/or rare aquatic species and enables biodiversity assessment without traditional sampling approaches, which are often labor-intensive. However, our understanding of the environmental factors that impact eDNA removal (i.e., how rapidly eDNA is removed from the water column by the combination of decay and physical removal) in flowing waters is limited. This limitation constrains predictions about the location and density of target organisms after positive detection. To address this question, we spiked Common Carp (Cyprinus carpio) eDNA into recirculating mesocosms (n = 24) under varying light (shaded versus open) and benthic substrate conditions (no substrate, bare substrate, and biofilm-colonized substrate). We then collected water samples from each mesocosm at four time points (40 min, 6 h, 18 h, and 48 h), and sequentially filtered the samples through 10, 1.0, and 0.2 µm filters to quantify removal rates for different eDNA particle sizes under varying light and substrate conditions. Combining all size classes, total eDNA removal rates were higher for mesocosms with biofilm-colonized substrate compared to those with no substrate or bare (i.e., no biofilm) substrate, which is consistent with previous findings linking biofilm colonization with increased eDNA removal and degradation. Additionally, when biofilm was present, light availability increased eDNA removal; eDNA levels fell below detection after 6-18 h for open mesocosms versus 18-48 h for shaded mesocosms. Among size classes, larger particles (>10 µm) were removed faster than small particles (1.0-0.2 µm). These results suggest that changes in the distribution of eDNA size classes over time (e.g., with downstream transport) and with differing environmental conditions could be used to predict the location of target organisms in flowing waters, which will advance the use of eDNA as a tool for species monitoring and management.

Structured Ethical Review for Wastewater-Based Testing.

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 the field blurred the boundary between measuring biomarkers for research activities and for pursuit of public health goals, both areas with well-established ethical frameworks. Currently, WBT practitioners do not employ a standardized ethical review process (or associated data management safeguards), introducing the potential for adverse outcomes for WBT professionals and community members. To address this deficiency, an interdisciplinary group 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 because of the common exemption of wastewater samples to human subject research considerations. This study retrospectively applied the set of questions to peer- reviewed published reports on SARS-CoV-2 monitoring campaigns covering the emergent phase of the pandemic from March 2020 to February 2022 (n=53). Overall, 43% of the responses to the questions were unable to be assessed because of lack of reported information. It is therefore hypothesized that a systematic framework would at a minimum improve the communication of key ethical considerations for the application of WBT. Consistent application of a standardized ethical review will also assist in developing an engaged practice of critically applying and updating approaches and techniques to reflect the concerns held by both those practicing and being monitored by WBT supported campaigns. Synopsis: Development of a structured ethical review facilitates retrospective analysis of published studies and drafted scenarios in the context of wastewater-based testing.

Simultaneous detection and quantification of multiple pathogen targets in wastewater.

Wastewater-based epidemiology has emerged as a critical tool for public health surveillance, building on decades of environmental surveillance work for pathogens such as poliovirus. Work to date has been limited to monitoring a single pathogen or small numbers of pathogens in targeted studies; however, few studies consider simultaneous quantitative analysis of a wide variety of pathogens, which could greatly increase the utility of wastewater surveillance. We developed a novel quantitative multi-pathogen surveillance approach (35 pathogen targets including bacteria, viruses, protozoa, and helminths) using TaqMan Array Cards (TAC) and applied the method on concentrated wastewater samples collected at four wastewater treatment plants in Atlanta, GA from February to October of 2020. From sewersheds serving approximately 2 million people, we detected a wide range of targets including many we expected to find in wastewater (e.g., enterotoxigenic E. coli and Giardia in 97% of 29 samples at stable concentrations) as well as unexpected targets including Strongyloides stercoralis (a human threadworm rarely observed in the USA). Other notable detections included SARS-CoV-2, but also several pathogen targets that are not commonly included in wastewater surveillance like Acanthamoeba spp., Balantidium coli, Entamoeba histolytica, astrovirus, norovirus, and sapovirus. Our data suggest broad utility in expanding the scope of enteric pathogen surveillance in wastewaters, with potential for application in a variety of settings where pathogen quantification in fecal waste streams can inform public health surveillance and selection of control measures to limit infections.

Show us the data: global COVID-19 wastewater monitoring efforts, equity, and gaps.

A year since the declaration of the global coronavirus disease 2019 (COVID-19) pandemic, there were over 110 million cases and 2.5 million deaths. Learning from methods to track community spread of other viruses such as poliovirus, environmental virologists and those in the wastewater-based epidemiology (WBE) field quickly adapted their existing methods to detect SARS-CoV-2 RNA in wastewater. Unlike COVID-19 case and mortality data, there was not a global dashboard to track wastewater monitoring of SARS-CoV-2 RNA worldwide. This study provides a 1-year review of the "COVIDPoops19" global dashboard of universities, sites, and countries monitoring SARS-CoV-2 RNA in wastewater. Methods to assemble the dashboard combined standard literature review, Google Form submissions, and daily, social media keyword searches. Over 200 universities, 1400 sites, and 55 countries with 59 dashboards monitored wastewater for SARS-CoV-2 RNA. However, monitoring was primarily in high-income countries (65%) with less access to this valuable tool in low- and middle-income countries (35%). Data were not widely shared publicly or accessible to researchers to further inform public health actions, perform meta-analysis, better coordinate, and determine equitable distribution of monitoring sites. For WBE to be used to its full potential during COVID-19 and beyond, show us the data.

Unveiling indicator, enteric, and respiratory viruses in aircraft lavatory wastewater using adsorption-extraction and Nanotrap® Microbiome A Particles workflows.

The effective detection of viruses in aircraft wastewater is crucial to establish surveillance programs for monitoring virus spread via aircraft passengers. This study aimed to compare the performance of two virus concentration workflows, adsorption-extraction (AE) and Nanotrap® Microbiome A Particles (NMAP), in detecting the prevalence and concentrations of 15 endogenous viruses including ssDNA, dsDNA, ssRNA in 24 aircraft lavatory wastewater samples. The viruses tested included two indicator viruses, four enteric viruses, and nine respiratory viruses. The results showed that cross-assembly phage (crAssphage), human polyomavirus (HPyV), rhinovirus A (RhV A), and rhinovirus B (RhV B) were detected in all wastewater samples using both workflows. However, enterovirus (EV), human norovirus GII (HNoV GII), human adenovirus (HAdV), bocavirus (BoV), parechovirus (PeV), epstein-barr virus (EBV). Influenza A virus (IAV), and respiratory syncytial virus B (RsV B) were infrequently detected by both workflows, and hepatitis A virus (HAV), influenza B virus (IBV), and respiratory syncytial virus B (RsV A) were not detected in any samples. The NMAP workflow had greater detection rates of RNA viruses (EV, PeV, and RsV B) than the AE workflow, while the AE workflow had greater detection rates of DNA viruses (HAdV, BoV, and EBV) than the NMAP workflow. The concentration of each virus was also analyzed, and the results showed that crAssphage had the highest mean concentration (6.76 log10 GC/12.5 mL) followed by HPyV (5.46 log10 GC/12.5 mL using the AE workflow, while the mean concentrations of enteric and respiratory viruses ranged from 2.48 to 3.63 log10 GC/12.5 mL. Using the NMAP workflow, the mean concentration of crAssphage was 5.18 log10 GC/12.5 mL and the mean concentration of HPyV was 4.20 log10 GC/12.5 mL, while mean concentrations of enteric and respiratory viruses ranged from 2.55 to 3.74 log10 GC/12.5 mL. Significantly higher (p < 0.05) mean concentrations of crAssphage and HPyV were observed when employing the AE workflow in comparison to the NMAP workflow. Conversely, the NMAP workflow yielded significantly greater (p < 0.05) concentrations of RhV A, and RhV B compared to the AE workflow. The findings of this study can aid in the selection of an appropriate concentration workflow for virus surveillance studies and contribute to the development of efficient virus detection methods.

Influence of membrane pore-size on the recovery of endogenous viruses from wastewater using an adsorption-extraction method.

The ongoing COVID-19 pandemic has emphasized the significance of wastewater surveillance in monitoring and tracking the spread of infectious diseases, including SARS-CoV-2. The wastewater surveillance approach detects genetic fragments from viruses in wastewater, which could provide an early warning of outbreaks in communities. In this study, we determined the concentrations of four types of endogenous viruses, including non-enveloped DNA (crAssphage and human adenovirus 40/41), non-enveloped RNA (enterovirus), and enveloped RNA (SARS-CoV-2) viruses, from wastewater samples using the adsorption-extraction (AE) method with electronegative HA membranes of different pore sizes (0.22, 0.45, and 0.80 µm). Our findings showed that the membrane with a pore size of 0.80 µm performed comparably to the membrane with a pore size of 0.45 µm for virus detection/quantitation (repeated measurement one-way ANOVA; p > 0.05). We also determined the recovery efficiencies of indigenous crAssphage and pepper mild mottle virus, which showed recovery efficiencies ranging from 50% to 94% and from 20% to 62%, respectively. Our results suggest that the use of larger pore size membranes may be beneficial for processing larger sample volumes, particularly for environmental waters containing low concentrations of viruses. This study offers valuable insights into the application of the AE method for virus recovery from wastewater, which is essential for monitoring and tracking infectious diseases in communities.

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.

Occurrence of multiple respiratory viruses in wastewater in Queensland, Australia: Potential for community disease surveillance.

The early warning and tracking of COVID-19 prevalence in the community provided by wastewater surveillance has highlighted its potential for much broader viral disease surveillance. In this proof-of-concept study, 46 wastewater samples from four wastewater treatment plants (WWTPs) in Queensland, Australia, were analyzed for the presence and abundance of 13 respiratory viruses, and the results were compared with reported clinical cases. The viruses were concentrated using the adsorption-extraction (AE) method, and extracted nucleic acids were analyzed using qPCR and RT-qPCR. Among the viruses tested, bocavirus (BoV), parechovirus (PeV), rhinovirus A (RhV A) and rhinovirus B (RhV B) were detected in all wastewater samples. All the tested viruses except influenza B virus (IBV) were detected in wastewater sample from at least one WWTP. BoV was detected with the greatest concentration (4.96-7.22 log10 GC/L), followed by Epstein-Barr virus (EBV) (4.08-6.46 log10 GC/L), RhV A (3.95-5.63 log10 GC/L), RhV B (3.74-5.61 log10 GC/L), and PeV (3.17-5.32 log10 GC/L). Influenza viruses and respiratory syncytial virus (RSV) are notifiable conditions in Queensland, allowing the gene copy (GC) concentrations to be compared with reported clinical cases. Significant correlations (ρ = 0.60, p < 0.01 for IAV and ρ = 0.53, p < 0.01 for RSV) were observed when pooled wastewater influenza A virus (IAV) and RSV log10 GC/L concentrations were compared to log10 clinical cases among the four WWTP catchments. The positive predictive value for the presence of IAV and RSV in wastewater was 97 % for both IAV and RSV clinical cases within the four WWTP catchments. The overall accuracy of wastewater analysis for predicting clinical cases of IAV and RSV was 97 and 90 %, respectively. This paper lends credibility to the application of wastewater surveillance to monitor respiratory viruses of various genomic characteristics, with potential uses for increased surveillance capabilities and as a tool in understanding the dynamics of disease circulation in the communities.

Comparative analysis of Adsorption-Extraction (AE) and Nanotrap® Magnetic Virus Particles (NMVP) workflows for the recovery of endogenous enveloped and non-enveloped viruses in wastewater.

In this study, two virus concentration methods, namely Adsorption-Extraction (AE) and Nanotrap® Magnetic Virus Particles (NMVP) along with commercially available extraction kits were used to quantify endogenous pepper mild mottle virus (PMMoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in nucleic acid extracted from 48 wastewater samples collected over six events from eight wastewater treatment plants (WWTPs). The main aim was to determine which workflow (i.e., concentration and extraction methods) produces greater concentrations of endogenous PMMoV and SARS-CoV-2 gene copies (GC) in comparison with each other. Turbidity and total suspended solids (TSS) of wastewater samples within and among the eight WWTPs were highly variable (41-385 NTU and 77-668 mg/L TSS). In 58 % of individual wastewater samples, the log10 GC concentrations of PMMoV were greater by NMVP workflow compared to AE workflow. Paired measurements of PMMoV GC/10 mL from AE and NMVP across all 48 wastewater samples were weakly correlated (r = 0.455, p = 0.001) and demonstrated a poor linear relationship (r2 = 0.207). The log10 GC concentrations of SARS-CoV-2 in 69 % of individual samples were greater by AE workflow compared to NMVP workflow. In contrast to PMMoV, the AE and NMVP derived SARS-CoV-2 GC counts were strongly correlated (r = 0.859, p < 0.001) and demonstrated a strong linear relationship (r2 = 0.738). In general, the PMMoV GC achieved by the NMVP workflow decreased with increasing turbidity, but the PMMoV GC by the AE workflow did not appear to be as sensitive to either turbidity or TSS levels. These findings suggest that wastewater sample turbidity or suspended solids concentration, and the intended target for analysis should be considered when validating an optimal workflow for wastewater surveillance of viruses.

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.

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.

Looking Forward: The Role of Academic Researchers in Building Sustainable Wastewater Surveillance Programs.

BACKGROUND: In just over 2 years, tracking the COVID-19 pandemic through wastewater surveillance advanced from early reports of successful SARS-CoV-2 RNA detection in untreated wastewater to implementation of programs in at least 60 countries. Early wastewater monitoring efforts primarily originated in research laboratories and are now transitioning into more formal surveillance programs run in commercial and public health laboratories. A major challenge in this progression has been to simultaneously optimize methods and build scientific consensus while implementing surveillance programs, particularly during the rapidly changing landscape of the pandemic. Translating wastewater surveillance results for effective use by public health agencies also remains a key objective for the field. OBJECTIVES: We examined the evolution of wastewater surveillance to identify model collaborations and effective partnerships that have created rapid and sustained success. We propose needed areas of research and key roles academic researchers can play in the framework of wastewater surveillance to aid in the transition from early monitoring efforts to more formalized programs within the public health system. DISCUSSION: Although wastewater surveillance has rapidly developed as a useful public health tool for tracking COVID-19, there remain technical challenges and open scientific questions that academic researchers are equipped to address. This includes validating methodology and backfilling important knowledge gaps, such as fate and transport of surveillance targets and epidemiological links to wastewater concentrations. Our experience in initiating and implementing wastewater surveillance programs in the United States has allowed us to reflect on key barriers and draw useful lessons on how to promote synergy between different areas of expertise. As wastewater surveillance programs are formalized, the working relationships developed between academic researchers, commercial and public health laboratories, and data users should promote knowledge co-development. We believe active involvement of academic researchers will contribute to building robust surveillance programs that will ultimately provide new insights into population health. https://doi.org/10.1289/EHP11519.

Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV-2 RNA in Wastewater.

We compared reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RT digital PCR (RT-dPCR) platforms for the trace detection of SARS-CoV-2 RNA in low-prevalence COVID-19 locations in Queensland, Australia, using CDC N1 and CDC N2 assays. The assay limit of detection (ALOD), PCR inhibition rates, and performance characteristics of each assay, along with the positivity rates with the RT-qPCR and RT-dPCR platforms, were evaluated by seeding known concentrations of exogenous SARS-CoV-2 in wastewater. The ALODs using RT-dPCR were approximately 2-5 times lower than those using RT-qPCR. During sample processing, the endogenous (n = 96) and exogenous (n = 24) SARS-CoV-2 wastewater samples were separated, and RNA was extracted from both wastewater eluates and pellets (solids). The RT-dPCR platform demonstrated a detection rate significantly greater than that of RT-qPCR for the CDC N1 and CDC N2 assays in the eluate (N1, p = 0.0029; N2, p = 0.0003) and pellet (N1, p = 0.0015; N2, p = 0.0067) samples. The positivity results also indicated that for the analysis of SARS-CoV-2 RNA in wastewater, including the eluate and pellet samples may further increase the detection sensitivity using RT-dPCR.

Distribution of human fecal marker genes and their association with pathogenic viruses in untreated wastewater determined using quantitative PCR.

Quantitative microbial risk assessment (QMRA) of human health risks using human fecal marker genes (HFMGs) is an useful water quality management tool. To inform accurate QMRA analysis, generation of probability distribution functions for HFMGs, and reference pathogenic viruses can be improved by input of correlation and ratios based upon measurement of HFMGs and gene copies (GC) of pathogenic viruses in untreated wastewater. The concentrations of four HFMGs (Bacteroides HF183, Lachnospiraceae Lachno3, CrAssphage and pepper mild mottle virus (PMMoV)), and GC of three reference pathogenic viruses human adenovirus 40/41 (HAdV 40/41), human norovirus GI + GII HNoV GI + GII and enterovirus (EV) were measured in untreated wastewater samples collected over a period of 12 months from two wastewater treatment plants in Sydney, Australia using quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR). Over the course of the study, the GC of potential pathogenic viruses were 3-5 orders of magnitude lower than HFMGs in untreated wastewater. The GC of pathogenic viruses were highly variable over the course of the study, which contrasted with the concentrations of HFMGs that were quite stable with little variation observed within and between WWTPs. Among the HFMGs, HF183, CrAssphage and PMMoV correlated well with pathogenic virus GC, whereas weak or negative correlations were observed between Lachno3 and pathogenic virus GC. While the two assessed WWTPs had dissimilar population service sizes, the ratios between log10 transformed pathogenic virus GC and HFMGs demonstrated similar central tendency and variability for the same combinations between WWTP A and WWTP B with no difference between the WWTPs. This suggests the widespread presence of these HFMGs in both populations serviced by these two WWTPs. The observed correlation and ratios of HFMGs and GC of reference pathogenic viruses can contribute to improved QMRA of human health risks in environmental waters subject to fresh sewer overflows.