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.

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.

Decay of infectious adenovirus and coliphages in freshwater habitats is differentially affected by ambient sunlight and the presence of indigenous protozoa communities.

BACKGROUND: Sanitary quality of recreational waters worldwide is assessed using fecal indicator bacteria (FIB), such as Escherichia coli and enterococci. However, fate and transport characteristics of FIB in aquatic habitats can differ from those of viral pathogens which have been identified as main etiologic agents of recreational waterborne illness. Coliphages (bacteriophages infecting E. coli) are an attractive alternative to FIB because of their many morphological and structural similarities to viral pathogens. METHODS: In this in situ field study, we used a submersible aquatic mesocosm to compare decay characteristics of somatic and F+ coliphages to those of infectious human adenovirus 2 in a freshwater lake. In addition, we also evaluated the effect of ambient sunlight (and associated UV irradiation) and indigenous protozoan communities on decay of somatic and F+ coliphage, as well as infectious adenovirus. RESULTS: Our results show that decay of coliphages and adenovirus was similar (p = 0.0794), indicating that both of these bacteriophage groups are adequate surrogates for decay of human adenoviruses. Overall, after 8 days the greatest log10 reductions were observed when viruses were exposed to a combination of biotic and abiotic factors (2.92 ± 0.39, 4.48 ± 0.38, 3.40 ± 0.19 for somatic coliphages, F+ coliphages and adenovirus, respectively). Both, indigenous protozoa and ambient sunlight, were important contributors to decay of all three viruses, although the magnitude of that effect differed over time and across viral targets. CONCLUSIONS: While all viruses studied decayed significantly faster (p < 0.0001) when exposed to ambient sunlight, somatic coliphages were particularly susceptible to sunlight irradiation suggesting a potentially different mechanism of UV damage compared to F+ coliphages and adenoviruses. Presence of indigenous protozoan communities was also a significant contributor (p value range: 0.0016 to < 0.0001) to decay of coliphages and adenovirus suggesting that this rarely studied biotic factor is an important driver of viral reductions in freshwater aquatic habitats.

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.

A Constructed Wetland for Treatment of an Impacted Waterway and the Influence of Native Waterfowl on its Perceived Effectiveness.

A constructed, variable-flow treatment wetland was evaluated for its ability to reduce microbial loads from the Banklick Creek, an impacted recreational waterway in Northern Kentucky. For this study, levels of traditional (Escherichia coli and enterococci measured by culture and molecular techniques) and alternative fecal indicators (infectious somatic and F+ coliphage, Clostridium spp. and Clostridium perfringens by culture), potential pathogens (molecular signal of Campylobacter spp.) as well as various microbial source tracking (MST) markers (human fecal marker HF183 and avian fecal marker GFD) were monitored during the summer and early fall through five treatment stages within the Banklick Creek Wetland. No difference in concentrations of traditional or alternative fecal indicators were observed in any of the sites monitored. Microbial source tracking markers were employed to identify sources of fecal contamination within the wetland. Human marker HF183 concentrations at beginning stages of treatment were found to be significantly higher (P value range: 0.0016-0.0003) than levels at later stages. Conversely, at later stages of treatment where frequent bird activity was observed, Campylobacter and avian marker (GFD) signals were detected at significantly higher frequencies (P value range: 0.024 to <0.0001), and both signals were strongly correlated (P = 0.0001). Our study suggests constructed wetlands are an effective means for removal of microbial contamination in ambient waters, but reliance on general fecal indicators is not ideal for determining system efficacy or assessing appropriate remediation efforts.

A Spike Cocktail Approach to Improve Microbial Performance Monitoring for Water Reuse.

Water reuse, via either centralized treatment of traditional wastewater or decentralized treatment and on-site reuse, is becoming an increasingly important element of sustainable water management. Despite advances in waterborne pathogen detection methods, low and highly variable pathogen levels limit their utility for routine evaluation of health risks in water reuse systems. Therefore, there is a need to improve our understanding of the linkage between pathogens and more readily measured process indicators during treatment. This paper describes an approach for constructing spiking experiments to relate the behavior of viral, bacterial, and protozoan pathogens with relevant process indicators. General issues are reviewed, and the spiking protocol is applied as a case study example to improve microbial performance monitoring and health risk evaluation in a water reuse system. This approach provides a foundation for the development of novel approaches to improve real or near-real time performance monitoring of water recycling systems.

Biotic interactions and sunlight affect persistence of fecal indicator bacteria and microbial source tracking genetic markers in the upper Mississippi river.

The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (Escherichia coli and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An in situ mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (r(2), 0.96 to 0.98; P < 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence.

The comparison of decay rates of infectious SARS-CoV-2 and viral RNA in environmental waters and wastewater.

Understanding the decay characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater and ambient waters is important for multiple applications including assessment of risk of exposure associated with handling wastewater samples, public health risk associated with recreation in wastewater polluted ambient waters and better understanding and interpretation of wastewater-based epidemiology (WBE) results. We evaluated the decay rates of infectious SARS-CoV-2 and viral RNA in wastewater and ambient waters under temperature regimes representative of seasonal fluctuations. Infectious virus was seeded in autoclaved primary wastewater effluent, final dechlorinated wastewater effluent, lake water, and marine water at a final concentration of 6.26 ± 0.07 log10 plaque forming units per milliliter. Each suspension was incubated at either 4°, 25°, and 37 °C. Samples were initially collected on an hourly basis, then approximately every other day for 15 days. All samples were analyzed for infectious virus via a plaque assay using the Vero E6 cell line, and viral gene copy levels were quantified with the US CDC's N1 and N2 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. The infectious virus decayed significantly faster (p ≤ 0.0214) compared to viral RNA, which persisted for the duration of the study irrespective of the incubation conditions. The initial loss (within 15 min of seeding) as well as decay of infectious SARS-CoV-2 was significantly faster (p ≤ 0.0387) in primary treated wastewater compared to other water types, but viral RNA did not degrade appreciably in this matrix until day 15. Overall, temperature was the most important driver of decay, and after 24 h, no infectious SARS-CoV-2 was detected at 37 °C in any water type. Moreover, the CDC N2 gene assay target decayed significantly (p ≤ 0.0174) faster at elevated temperatures compared to CDC N1, which has important implications for RT-qPCR assay selection for WBE approach.

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.

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.

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.

Indigenous microbiota and habitat influence Escherichia coli survival more than sunlight in simulated aquatic environments.

The reported fate of Escherichia coli in the environment ranges from extended persistence to rapid decline. Incomplete understanding of factors that influence survival hinders risk assessment and modeling of the fate of fecal indicator bacteria (FIB) and pathogens. FIB persistence in subtropical aquatic environments was explored in outdoor mesocosms inoculated with five E. coli strains. The manipulated environmental factors were (i) presence or absence of indigenous microbiota (attained by natural, disinfected, and cycloheximide treatments), (ii) freshwater versus seawater, and (iii) water column versus sediment matrices. When indigenous microbes were removed (disinfected), E. coli concentrations decreased little despite exposure to sunlight. Conversely, under conditions that included the indigenous microbiota (natural), significantly greater declines in E. coli occurred regardless of the habitat. The presence of indigenous microbiota and matrix significantly influenced E. coli decline, but their relative importance differed in freshwater versus seawater. Cycloheximide, which inhibits protein synthesis in eukaryotes, significantly diminished the magnitude of E. coli decline in water but not in sediments. The inactivation of protozoa and bacterial competitors (disinfected) caused a greater decline in E. coli than cycloheximide alone in water and sediments. These results indicate that the autochthonous microbiota are an important contributor to the decline of E. coli in fresh and seawater subtropical systems, but their relative contribution is habitat dependent. This work advances our understanding of how interactions with autochthonous microbiota influence the fate of E. coli in aquatic environments and provides the framework for studies of the ecology of enteric pathogens and other allochthonous bacteria in similar environments.

Differential decay of enterococci and Escherichia coli originating from two fecal pollution sources.

Using in situ subtropical aquatic mesocosms, fecal source (cattle manure versus sewage) was shown to be the most important contributor to differential loss in viability of fecal indicator bacteria (FIB), specifically enterococci in freshwater and Escherichia coli in marine habitats. In this study, sunlight exposure and indigenous aquatic microbiota were also important contributors, whose effects on FIB also differed between water types.

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.

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.

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.

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.

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.