Comparison of adsorption-extraction (AE) workflows for improved measurements of viral and bacterial nucleic acid in untreated wastewater.

The lack of standardized methods and large differences in virus concentration and extraction workflows have hampered Severe Acute Respiratory Syndrome (SARS-CoV-2) wastewater surveillance and data reporting practices. Numerous studies have shown that adsorption-extraction (AE) method holds promise, yet several uncertainties remain regarding the optimal AE workflow. Several procedural components may influence the recovered concentrations of target nucleic acid, including membrane types, homogenization instruments, speed and duration, and lysis buffer. In this study, 42 different AE workflows that varied these components were compared to determine the optimal workflow by quantifying endogenous SARS-CoV-2, human adenovirus 40/41 (HAdV 40/41), and a bacterial marker gene of fecal contamination (Bacteroides HF183). Our findings suggest that the workflow chosen had a significant impact on SARS-CoV-2 concentrations, whereas it had minimal impact on HF183 and no effect on HAdV 40/41 concentrations. When comparing individual components in a workflow, such as membrane type (MF-Millipore™ 0.45 µm MCE vs. Isopore™ 0.40 µm), we found that they had no impact on SARS-CoV-2, HAdV 40/41, and HF183 concentrations. This suggests that at least some consumables and equipment are interchangeable. Buffer PM1 + TRIzol-based workflows yielded higher concentrations of SARS-CoV-2 than other workflows. HF183 concentrations were higher in workflows without chloroform. Similarly, higher homogenization speeds (5000-10,000 rpm) led to increased concentrations of SARS-CoV-2 and HF183 but had no effect on HAdV 40/41. Our findings indicate that minor enhancements to the AE workflow can improve the recovery of viruses and bacteria from the wastewater, leading to improved outcomes from wastewater surveillance efforts.

Assessing the nucleic acid decay of human wastewater markers and enteric viruses in estuarine waters in Sydney, Australia.

This research investigated the in-situ decay rates of four human wastewater-associated markers (Bacteroides HF183 (HF183), Lachnospiraceae Lachno3 (Lachno3), cross-assembling phage (crAssphage), pepper mild mottle virus (PMMoV) and three enteric viruses (human adenovirus 40/41 (HAdV 40/41), enterovirus (EV) and human norovirus GII (HNoV GII) in two estuarine water environments (Davidson Park (DP) and Hen and Chicken Bay (HCB) in temperate Sydney, NSW, Australia, employing qPCR and RT-qPCR assays. The study also aimed to compare decay rates observed in mesocosms with previously published laboratory microcosms, providing insights into the persistence of markers and viruses in estuarine environments. Results indicated varying decay rates between DP and HCB mesocosms, with HF183 exhibiting relatively faster decay rates compared to other markers and enteric viruses in sunlight and dark mesocosms. In DP mesocosms, HF183 decayed the fastest, contrasting with PMMoV, which exhibited the slowest. Sunlight induced higher decay rates for all markers and viruses in DP mesocosms. In HCB sunlight mesocosms, HF183 nucleic acid decayed most rapidly compared to other markers and enteric viruses. In dark mesocosms, crAssphage showed the fastest decay, while PMMoV decayed at the slowest rate in both sunlight and dark mesocosms. Comparisons with laboratory microcosms revealed faster decay of markers and enteric viruses in laboratory microcosms than the mesocosms, except for crAssphage and HAdV 40/41 in dark, and PMMoV in sunlight mesocosms. The study concludes that decay rates of markers and enteric viruses vary between estuarine mesocosms, emphasizing the impact of sunlight exposure, which was potentially influenced by the elevated turbidity at HCB estuarine waters. The generated decay rates contribute valuable insights for establishing site-specific risk-based thresholds of human wastewater-associated markers.

Comparing the decay of human wastewater-associated markers and enteric viruses in laboratory microcosms simulating estuarine waters in a temperate climatic zone using qPCR/RT-qPCR assays.

This study investigated the decay rates of wastewater-associated markers and enteric viruses in laboratory microcosms mimicking estuarine water environments in temperate Sydney, NSW, Australia using qPCR and RT-qPCR assays. The results demonstrated the reduction in concentrations of Bacteroides HF183, Lachnospiraceae Lachno3, cross-assembly phage (crAssphage), pepper mild mottle virus (PMMoV), human adenovirus (HAdV 40/41), and enterovirus (EV) over a span of 42 days under spring/summer temperatures, presence/absence of microbiota, and different light conditions. The study found that HF183, Lachno3, crAssphage, PMMoV, HAdV 40/41, and EV exhibited varying decay rates depending on the experimental conditions. The average T90 values ranged from a few days to several months, indicating the rapid decay or prolonged persistence of these markers and enteric viruses in the estuarine environment. Furthermore, the study examined the effects of indigenous microbiota and spring/summer temperatures on wastewater-associated markers and enteric viruses decay rates. It was found that the presence of microbiota and temperature significantly influenced the decay rates of HF183 and PMMoV. Additionally, the study compared the effects of artificial sunlight and spring/summer temperatures on marker decay rates. Bacterial markers decayed faster than viral markers, although among viral markers crAssphage decay rates were relatively faster when compared to PMMoV. The exposure to artificial sunlight significantly accelerated the decay rates of bacterial markers, viral markers, and enteric viruses. Temperature also had an impact on the decay rates of Lachno3, crAssphage, and HAdV 40/41. In conclusion, this study provides valuable insights into the decay rates of wastewater-associated markers and enteric viruses under different experimental conditions that mimicked temperate environmental conditions. The findings contribute to our understanding of the fate and persistence of these markers in the environment which is crucial for assessing and managing risks from contamination by untreated human wastewater.

Improved virus concentration methods for wash waters from decontamination of permeable and non-permeable surfaces.

Surface decontamination is a method of using wash water to decontaminated surfaces preventing transmission of biological contaminants that can pose potential health risks to responders and the public. However, the risks associated with handling used wash water are largely unknown due to the lack of effective methodology to screen for pathogenic microorganisms present in these samples, especially viral pathogens. This study adapted the dead-end hollow-fiber ultrafiltration (D-HFUF) system to wash waters, including a separate procedure for recovering particle attached viruses. Simulated wash water was created using dechlorinated tap water containing a mild surfactant (0.05 % Tween 80). To determine virus recovery efficiencies, measured amounts of somatic and F+ coliphage were spiked into 2-liter volumes of wash water under the following scenarios: (1) wash water was amended with a measured amount of sterile river sediment with no sediment separation prior to filter concentration; or (2) sediment added to wash water was allowed to settle prior to filter concentrating clarified liquid portions, while precipitated sediment was subjected to viral extraction techniques to recover particle attached virus; and (3) the optimized method was deployed on non-porous and porous surfaces to simulate a decontamination clean-up event. Separation of sediment prior to D-HFUF significantly increased recovery of coliphages, (P = <0.0001) versus filtration of sediment and liquids simultaneously. A tryptic soy broth (TSB) elution solution was significantly more effective (P = ≤0.010) for recovery of both somatic and F+ coliphage, (108 ± 9 % and 92 ± 9 %, respectively), compared to elution buffers containing various surfactants (sodium hexametaphosphate, Tween 80) for recovering particle attached virus. Simulating a biocontaminate clean-up event (using the optimized sediment separation and elution protocol) resulted in coliphage recoveries of 75-96 % (permeable surface) and 71-92 % (non-permeable surface). This procedure can be used to effectively detect viruses in used wash waters aiding in reducing risks to human health during site decontamination.

The Effect of Protozoa Indigenous to Lakewater and Wastewater on Decay of Fecal Indicator Bacteria and Coliphage.

Fecal indicator bacteria (FIB: Escherichia coli and enterococci) are used to assess recreational water quality. Viral indicators (i.e., somatic and F+ coliphage), could improve the prediction of viral pathogens in recreational waters, however, the impact of environmental factors, including the effect of predatory protozoa source, on their survival in water is poorly understood. We investigated the effect of lakewater or wastewater protozoa, on the decay (decreasing concentrations over time) of culturable FIB and coliphages under sunlight and shaded conditions. FIB decay was generally greater than the coliphages and was more rapid when indicators were exposed to lake vs. wastewater protozoa. F+ coliphage decay was the least affected by experimental variables. Somatic coliphage decayed fastest in the presence of wastewater protozoa and sunlight, though their decay under shaded conditions was-10-fold less than F+ after 14 days. The protozoa source consistently contributed significantly to the decay of FIB, and somatic, though not the F+ coliphage. Sunlight generally accelerated decay, and shade reduced somatic coliphage decay to the lowest level among all the indicators. Differential responses of FIB, somatic, and F+ coliphages to environmental factors support the need for studies that address the relationship between the decay of coliphages and viral pathogens under environmentally relevant conditions.

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.

Assessment of two volumetrically different concentration approaches to improve sensitivities for SARS-CoV-2 detection during wastewater monitoring.

Wastewater monitoring for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the virus responsible for the global coronavirus disease 2019 (COVID-19) pandemic, has highlighted the need for methodologies capable of assessing viral prevalence during periods of low population infection. To address this need, two volumetrically different, methodologically similar concentration approaches were compared for their abilities to detect viral nucleic acid and infectious SARS-CoV-2 signal from primary influent samples. For Method 1, 2 L of SARS-CoV-2 seeded wastewater was evaluated using a dead-end hollow fiber ultrafilter (D-HFUF) for primary concentration, followed by the CP Select™ for secondary concentration. For Method 2, 100 mL of SARS-CoV-2 seeded wastewater was evaluated using the CP Select™ procedure. Following D-HFUF concentration (Method 1), significantly lower levels of infectious SARS-CoV-2 were lost (P value range: 0.0398-0.0027) compared to viral gene copy (GC) levels detected by the US Centers for Disease Control (CDC) N1 and N2 reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. Subsamples at different steps in the concentration process were also taken to better characterize the losses of SARS-CoV-2 during the concentration process. During the centrifugation step (prior to CP Select™ concentration), significantly higher losses (P value range: 0.0003 to <0.0001) occurred for SARS-CoV-2 GC levels compared to infectious virus for Method 1, while between the methods, significantly higher infectious viral losses were observed for Method 2 (P = 0.0002). When analyzing overall recovery of endogenous SARS-CoV-2 in wastewater samples, application of Method 1 improved assay sensitivities (P = <0.0001) compared with Method 2; this was especially evident during periods of lower COVID-19 case rates within the sewershed. This study describes a method which can successfully concentrate infectious SARS-CoV-2 and viral RNA from wastewater. Moreover, we demonstrated that large volume wastewater concentration provides additional sensitivity needed to improve SARS-CoV-2 detection, especially during low levels of community disease prevalence.

Bacterial and viral fecal indicator predictive modeling at three Great Lakes recreational beach sites.

Coliphage are viruses that infect Escherichia coli (E. coli) and may indicate the presence of enteric viral pathogens in recreational waters. There is an increasing interest in using these viruses for water quality monitoring and forecasting; however, the ability to use statistical models to predict the concentrations of coliphage, as often done for cultured fecal indicator bacteria (FIB) such as enterococci and E. coli, has not been widely assessed. The same can be said for FIB genetic markers measured using quantitative polymerase chain reaction (qPCR) methods. Here we institute least-angle regression (LARS) modeling of previously published concentrations of cultured FIB (E. coli, enterococci) and coliphage (F+, somatic), along with newly reported genetic concentrations measured via qPCR for E. coli, enterococci, and general Bacteroidales. We develop site-specific models from measures taken at three beach sites on the Great Lakes (Grant Park, South Milwaukee, WI; Edgewater Beach, Cleveland, OH; Washington Park, Michigan City, IN) to investigate the efficacy of a statistical predictive modeling approach. Microbial indicator concentrations were measured in composite water samples collected five days per week over a beach season (∼15 weeks). Model predictive performance (cross-validated standardized root mean squared error of prediction [SRMSEP] and R2PRED) were examined for seven microbial indicators (using log10 concentrations) and water/beach parameters collected concurrently with water samples. Highest predictive performance was seen for qPCR-based enterococci and Bacteroidales models, with F+ coliphage consistently yielding poor performing models. Influential covariates varied by microbial indicator and site. Antecedent rainfall, bird abundance, wave height, and wind speed/direction were most influential across all models. Findings suggest that some fecal indicators may be more suitable for water quality forecasting than others at Great Lakes beaches.

RT-qPCR and ATOPlex sequencing for the sensitive detection of SARS-CoV-2 RNA for wastewater surveillance.

During 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 detect and quantify SARS-CoV-2 RNA in wastewater, while monitoring viral genome mutations requires separate approaches such as deep sequencing. A high throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique has shown promise in detecting variants of concern (VOC) while also providing virus quantitation data. However, detection sensitivities of both RT-qPCR and sequencing can be impacted through losses occurring during sample handling, virus concentration, nucleic acid extraction, and RT-qPCR. Therefore, process limit of detection (PLOD) assessments are required to estimate the gene copies of target molecule to attain specific probability of detection. In this study, we compare the PLOD of four RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab) for detection of SARS-CoV-2 to that of ATOPlex sequencing by seeding known concentrations of gamma-irradiated SARS-CoV-2 into wastewater. Results suggest that among the RT-qPCR assays, US CDC N1 was the most sensitive, especially at lower SARS-CoV-2 seed levels. However, when results from all RT-qPCR assays were combined, it resulted in greater detection rates than individual assays, suggesting that application of multiple assays is better suited for the trace detection of SARS-CoV-2 from wastewater samples. Furthermore, while ATOPlex offers a promising approach to SARS-CoV-2 wastewater surveillance, this approach appears to be less sensitive compared to RT-qPCR under the experimental conditions of this study, and may require further refinements. Nonetheless, the combination of RT-qPCR and ATOPlex may be a powerful tool to simultaneously detect/quantify SARS-CoV-2 RNA and monitor emerging VOC in wastewater samples.

Effectiveness of two wastewater disinfection strategies for the removal of fecal indicator bacteria, bacteriophage, and enteric viral pathogens concentrated using dead-end hollow fiber ultrafiltration (D-HFUF).

Primary influent and final effluent samples were collected from wastewater treatment plants using either chlorination or ultraviolet (UV) disinfection biweekly for one year. Paired measurements were determined for fecal indicator bacteria (Escherichia coli and enterococci), cultivated bacteriophages (somatic, F+, and CB-390 coliphage and GB-124 Bacteroides phage), human-associated viral markers (human polyomavirus [HPyV] and crAssphage), enteric pathogens (adenovirus, noroviruses genogroups I and II) as well as total infectious enteric virus. To increase the probability of detecting low concentration targets, both primary (10L) and final effluent wastewater samples (40-100 L) were concentrated using a dead-end hollow-fiber ultrafilter (D-HFUF). Despite seasonal temperature fluctuations, concentration shifts of FIB, bacteriophages, human-associated viruses, and viral pathogens measured in primary influent samples were minimal, while levels of infectious enteric virus were significantly higher in the spring and fall (P range: 0.0003-0.0409). FIB levels measured in primary influents were 1-2 log10 higher than bacteriophage, human-associated viral markers (except crAssphage) and viral pathogens measured. FIB displayed the greatest sensitivity to chlorine disinfection, while crAssphage, adenoviruses and infectious enteric viruses were significantly less sensitive (P ≤ 0.0096). During UV treatment, bacteriophages F+ and GB-124 were the most resistant of the culturable viruses measured (P ≤ 0.001), while crAssphage were the most resistant (P ≤ 0.0124) overall. When UV lamps were inactive, infectious enteric viruses were significantly more resilient to upstream treatment processes than all other targets measured (P ≤ 0.0257). Similar to infectious enteric viruses and adenoviruses; GB-124, F+, and crAssphages displayed the highest resistance to UV irradiation, signaling a potential applicability as pathogen surrogates in these systems. The use of D-HFUF enhanced the ability to estimate removal of viruses through wastewater treatment, with the expectation that future applications of this method will be used to better elucidate viral behavior within these systems.

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.

Comparative analysis of rapid concentration methods for the recovery of SARS-CoV-2 and quantification of human enteric viruses and a sewage-associated marker gene in untreated wastewater.

To support public-health-related disease surveillance and monitoring, it is crucial to concentrate both enveloped and non-enveloped viruses from domestic wastewater. To date, most concentration methods were developed for non-enveloped viruses, and limited studies have directly compared the recovery efficiency of both types of viruses. In this study, the effectiveness of two different concentration methods (Concentrating pipette (CP) method and an adsorption-extraction (AE) method amended with MgCl2) were evaluated for untreated wastewater matrices using three different viruses (SARS-CoV-2 (seeded), human adenovirus 40/41 (HAdV 40/41), and enterovirus (EV)) and a wastewater-associated bacterial marker gene targeting Lachnospiraceae (Lachno3). For SARS-CoV-2, the estimated mean recovery efficiencies were significantly greater by as much as 5.46 times, using the CP method than the AE method amended with MgCl2. SARS-CoV-2 RNA recovery was greater for samples with higher titer seeds regardless of the method, and the estimated mean recovery efficiencies using the CP method were 25.1 ± 11% across ten WWTPs when wastewater samples were seeded with 5 × 104 gene copies (GC) of SARS-CoV-2. Meanwhile, the AE method yielded significantly greater concentrations of indigenous HAdV 40/41 and Lachno3 from wastewater compared to the CP method. Finally, no significant differences in indigenous EV concentrations were identified in comparing the AE and CP methods. These data indicate that the most effective concentration method varies by microbial analyte and that the priorities of the surveillance or monitoring program should be considered when choosing the concentration method.

Performance evaluation of a dead-end hollowfiber ultrafiltration method for enumeration of somatic and F+ coliphage from recreational waters.

Dead-end hollow fiber ultrafiltration combined with a single agar layer assay (D-HFUF-SAL) has potential use in the assessment of sanitary quality of recreational waters through enumeration of coliphage counts as measures of fecal contamination. However, information on applicability across a broad range of sites and water types is limited. Here, we tested the performance of D-HFUF-SAL on 49 marine and freshwater samples. Effect of method used to titer the spiking suspension (SAL versus double agar layer [DAL]) on percent recovery was also evaluated. Average somatic coliphage recovery (72 % ± 27) was significantly higher (p < 0.0001) compared to F+ (53 % ± 19). This was more pronounced for marine (p ≤ 0.0001) compared to freshwaters (p = 0.0134). Neither method affected somatic coliphage, but DAL (28 % ± 12) significantly (p < 0.0001) underestimated F + coliphage recoveries compared to SAL (53 % ± 19). Overall, results indicate that, while D-HFUF-SAL performed well over a wide variety of water types, F + coliphage recoveries were significantly reduced for marine waters suggesting that some components unique to this habitat may interfere with the assay performance. More importantly, our findings indicate that choice of spike titer method merits careful consideration since it may under-estimate method percent recovery.

Development of a large volume concentration method for recovery of coronavirus from wastewater.

Levels of severe acute respiratory coronavirus type 2 (SARS CoV 2) RNA in wastewater could act as an effective means to monitor coronavirus disease 2019 (COVID-19) within communities. However, current methods used to detect SARS CoV 2 RNA in wastewater are limited in their ability to process sufficient volumes of source material, inhibiting our ability to assess viral load. Typically, viruses are concentrated from large liquid volumes using two stage concentration, primary and secondary. Here, we evaluated a dead-end hollow fiber ultrafilter (D-HFUF) for primary concentration, followed by the CP Select™ for secondary concentration from 2 L volumes of primary treated wastewater. Various amendments to each concentration procedure were investigated to optimally recover seeded OC43 (betacoronavirus) from wastewater. During primary concentration, the D-HFUF recovered 69 ± 18% (n = 29) of spiked OC43 from 2 L of wastewater. For secondary concentration, the CP Select™ system using the Wastewater Application settings was capable of processing 100 mL volumes of primary filter eluates in <25 min. A hand-driven syringe elution proved to be significantly superior (p = 0.0299) to the CP Select™ elution for recovering OC43 from filter eluates, 48 ± 2% compared to 31 ± 3%, respectively. For the complete method (primary and secondary concentration combined), the D-HFUF and CP select/syringe elution achieved overall 22 ± 4% recovery of spiked OC43 through (n = 8) replicate filters. Given the lack of available standardized methodology confounded by the inherent limitations of relying on viral RNA for wastewater surveillance of SARS CoV 2, it is important to acknowledge these challenges when interpreting this data to estimate community infection rates. However, the development of methods that can substantially increase sample volumes will likely allow for reporting of quantifiable viral data for wastewater surveillance, equipping public health officials with information necessary to better estimate community infection rates.

Intraday variability of indicator and pathogenic viruses in 1-h and 24-h composite wastewater samples: Implications for wastewater-based epidemiology.

We monitored the concentration of indicator viruses crAssphage and pepper mild mottle virus (PMMoV) and human pathogen adenovirus (HAdV) in influent from a wastewater treatment plant in Brisbane, Australia in 1-h and 24-h composite samples. Over three days of sampling, the mean concentration of crAssphage gene copies (GC)/mL in 24-h composite samples did not differ significantly (p = 0.72-0.92), while for PMMoV GC/mL (p value range: 0.0002-0.0321) and HAdV GC/mL (p value range: 0.0028-0.0068) significant differences in concentrations were observed on one day of sampling compared to the other two. For all three viruses, the variation observed in 1-h composite samples was greater than the variation observed in 24-h composite samples. For crAssphage, in 54.1% of 1-h composite samples, the concentration was less than that observed in 24-h composite samples; whereas for PMMoV and HAdV the concentration was less in 79.2 and 70.9% of 1-h composite samples, respectively, compared to the relevant 24-h composite samples. Similarly, the concentration of crAssphage in 1-h compared to 24-h composite samples did not differ (p = 0.1082) while the concentrations of PMMoV (p < 0.0001) and HAdV (p < 0.0001) in 1-h composite samples were significantly different from 24-h composite samples. These results suggest that 24-h composite samples offer increased analytical sensitivity and decreased variability compared to 1-h composite samples when monitoring wastewater, especially for pathogenic viruses with low infection rates within a community. Thus, for wastewater-based epidemiology applications, 24-h composite samples are less likely to produce false negative results and erroneous public health information.

Critical review of methods for isothermal amplification of nucleic acids for environmental analysis.

The past 30 years have seen the emergence and proliferation of isothermal amplification methods (IAMs) for rapid, sensitive detection and quantification of nucleic acids in a variety of sample types. These methods share dependence on primers and probes with quantitative PCR, but they differ in the specific enzymes and instruments employed, and are frequently conducted in a binary, rather than quantitative format. IAMs typically rely on simpler instruments than PCR analyses due to the maintenance of a single temperature throughout the amplification reaction, which could facilitate deployment of IAMs in a variety of environmental and field settings. This review summarizes the mechanisms of the most common IAM methods and their use in studies of pathogens, harmful algae and fecal indicators in environmental waters, feces, wastewater, reclaimed water, and tissues of aquatic animals. Performance metrics of sensitivity, specificity and limit of detection are highlighted, and the potential for use in monitoring and regulatory contexts is discussed.

Antibiotic-Resistant Enterococcus Species in Marine Habitats: A Review.

Antibiotic-resistant Enterococcus (ARE) are among leading causes of nosocomial infections worldwide. Enterococcus spp. are ubiquitous in sewage, which can contaminate surface waters via many pathways, providing a route of exposure for humans. This review focuses on ARE in marine and estuarine habitats, including marine animals. Phylogenetic confirmation of the genus Enterococcus and intermediate or full resistance to clinically relevant antibiotics were inclusion criteria. The proportion of resistant isolates varied greatly among antibiotics, for example, 24.2% for ampicillin and 2.4% for vancomycin. The water column contained the highest proportion of ARE observations (18.8%), followed by animal feces and tissues (14.8%), sediment (9.4%), and sand (2.0%). The proportion of multidrug-resistant isolates was the greatest in animal tissue and fecal samples, followed by water and sediments. This review indicates that clinically relevant ARE are present in marine/estuarine habitats and that animals may be important reservoirs.

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.

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.

Viral and Bacterial Fecal Indicators in Untreated Wastewater across the Contiguous United States Exhibit Geospatial Trends.

Cultivated fecal indicator bacteria such as Escherichia coli and enterococci are typically used to assess the sanitary quality of recreational waters. However, these indicators suffer from several limitations, such as the length of time needed to obtain results and the fact that they are commensal inhabitants of the gastrointestinal tract of many animals and have fate and transport characteristics dissimilar to pathogenic viruses. Numerous emerging technologies that offer same-day water quality results or pollution source information or that more closely mimic persistence patterns of disease-causing pathogens that may improve water quality management are now available, but data detailing geospatial trends in wastewater across the United States are sparse. We report geospatial trends of cultivated bacteriophage (somatic, F+, and total coliphages and GB-124 phage), as well as genetic markers targeting polyomavirus, enterococci, E. coli, Bacteroidetes, and human-associated Bacteroides spp. (HF183/BacR287 and HumM2) in 49 primary influent sewage samples collected from facilities across the contiguous United States. Samples were selected from rural and urban facilities spanning broad latitude, longitude, elevation, and air temperature gradients by using a geographic information system stratified random site selection procedure. Most indicators in sewage demonstrated a remarkable similarity in concentration regardless of location. However, some exhibited predictable shifts in concentration based on either facility elevation or local air temperature. Geospatial patterns identified in this study, or the absence of such patterns, may have several impacts on the direction of future water quality management research, as well as the selection of alternative metrics to estimate sewage pollution on a national scale.IMPORTANCE This study provides multiple insights to consider for the application of bacterial and viral indicators in sewage to surface water quality monitoring across the contiguous United States, ranging from method selection considerations to future research directions. Systematic testing of a large collection of sewage samples confirmed that crAssphage genetic markers occur at a higher average concentration than key human-associated Bacteroides spp. on a national scale. Geospatial testing also suggested that some methods may be more suitable than others for widespread implementation. Nationwide characterization of indicator geospatial trends in untreated sewage represents an important step toward the validation of these newer methods for future water quality monitoring applications. In addition, the large paired-measurement data set reported here affords the opportunity to conduct a range of secondary analyses, such as the generation of new or updated quantitative microbial risk assessment models used to estimate public health risk.