Virus surrogates throughout a full-scale advanced water reuse system.

Water reuse as an alternative water supply is increasing throughout the world due to water stress and scarcity; however, there are no standard practices for monitoring virus pathogens in such systems. This study aimed to identify suitable surrogates for virus fate, transport, and removal throughout a water reuse scheme. Various microbial targets (11 viruses, two phage, and three bacteria) were monitored using molecular and culture methods across all treatment stages in a wastewater reclamation facility and advanced water treatment facility. Criteria were established for identifying suitable surrogates, which included reliable detection, observable fate and transport, calculable log-reduction values (LRVs), correlations with other targets, and various morphological types. In total, five viruses (PMMoV, AiV, GII NoV, AdV, FRNA GII) met these stringent criteria and were suggested as potential virus surrogates. These surrogates enabled successful comparison of assigned versus actual LRVs throughout a water reuse scheme. Results suggest that virus pathogens are effectively removed throughout water reuse treatment and the suggested surrogates can be utilized for monitoring treatment performance and ensuring public health safety. This study provides a framework that water utilities across the world can reference for establishing virus monitoring practices.

Community-Scale Wastewater Surveillance of Candida auris during an Ongoing Outbreak in Southern Nevada.

Candida auris is an opportunistic fungal pathogen and an emerging global public health threat, given its high mortality among infected individuals, antifungal resistance, and persistence in healthcare environments. This study explored the applicability of wastewater surveillance for C. auris in a metropolitan area with reported outbreaks across multiple healthcare facilities. Influent or primary effluent samples were collected over 10 weeks from seven sewersheds in Southern Nevada. Pelleted solids were analyzed using an adapted quantitative polymerase chain reaction (qPCR) assay targeting the ITS2 region of the C. auris genome. Positive detection was observed in 72 of 91 samples (79%), with higher detection frequencies in sewersheds serving healthcare facilities involved in the outbreak (94 vs 20% sample positivity). Influent wastewater concentrations ranged from 2.8 to 5.7 log10 gene copies per liter (gc/L), and primary clarification achieved an average log reduction value (LRV) of 1.24 ± 0.34. Presumptive negative surface water and wastewater controls were non-detect. These results demonstrate that wastewater surveillance may assist in tracking the spread of C. auris and serve as an early warning tool for public health action. These findings provide the foundation for future application of wastewater-based epidemiology (WBE) to community- or facility-level surveillance of C. auris and other high consequence, healthcare-associated infectious agents.

SARS-CoV-2 genetic material is removed during municipal wastewater treatment and is undetectable after advanced treatment.

The aim of this study was to establish whether SARS-CoV-2 genetic material is detectable after municipal wastewater treatment and to verify its expected removal from purified water that is reclaimed for potable reuse. Viral loads of SARS-CoV-2 (N1 and N2 genes) were monitored in raw influent wastewater (sewage) entering a water reclamation facility and in subsequent advanced treatment. Despite the large viral RNA load in raw sewage during peak COVID-19 outbreaks, substantial amounts of SARS-CoV-2 genetic material were removed during the conventional wastewater treatment process. Further, SARS-CoV-2 genetic material was undetectable after advanced purification. This confirms that potable reuse is resilient against high viral loads which are expected results given the advanced degree of wastewater and water treatment. Findings from this study may enhance public perception of the safety of potable water reuse; however, it should also be noted that studies to date worldwide indicate no evidence of SARS-CoV-2 transmission via water, and the CDC does not consider fecal waste or wastewaters as a source of exposure.

Variant-specific SARS-CoV-2 shedding rates in wastewater.

Previous studies show that SARS-CoV-2 waste shedding rates vary by community and are influenced by multiple factors; however, differences in shedding rates across multiple variants have yet to be evaluated. The purpose of this work is to build on previous research that evaluated waste shedding rates for early SARS-CoV-2 and the Delta variant, and update population level waste shedding rates for the more-recent Omicron variant in six communities. Mean SARS-CoV-2 waste shedding rates were found to increase with the predominance of the Delta variant and subsequently decrease with Omicron infections. Interestingly, the Delta stage had the highest mean shedding rates and was associated with the most severe disease symptoms reported in other clinical studies, while Omicron, exhibiting reduced symptoms, had the lowest mean shedding rates. Additionally, shedding rates were most consistent across communities during the Omicron stage. This is the first paper to identify waste shedding rates specific to the Omicron variant and fills a knowledge gap critical to disease prevalence modeling.

Wastewater-Based Epidemiology Mitigates COVID-19 Outbreaks at a Food Processing Facility near the Mexico-U.S. Border-November 2020-March 2022.

Background: Wastewater-based epidemiology (WBE) has the potential to inform activities to contain infectious disease outbreaks in both the public and private sectors. Although WBE for SARS-CoV-2 has shown promise over short time intervals, no other groups have evaluated how a public-private partnership could influence disease spread through public health action over time. The aim of this study was to characterize and assess the application of WBE to inform public health response and contain COVID-19 infections in a food processing facility. Methods: Over the period November 2020-March 2022, wastewater in an Arizona food processing facility was monitored for the presence of SARS-CoV-2 using Real-Time Quantitative PCR. Upon positive detection, partners discussed public health intervention strategies, including infection control reinforcement, antigen testing, and vaccination. Results: SARS-CoV-2 RNA was detected on 18 of 205 days in which wastewater was sampled and analyzed (8.8%): seven during Wild-type predominance and 11 during Omicron-variant predominance. All detections triggered the reinforcement of infection control guidelines. In five of the 18 events, active antigen testing identified asymptomatic workers. Conclusions: These steps heightened awareness to refine infection control protocols and averted possible transmission events during periods where detection occurred. This public-private partnership has potentially decreased human illness and economic loss during the COVID-19 pandemic.

Population level SARS-CoV-2 fecal shedding rates determined via wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) has been utilized as an early warning tool to anticipate disease outbreaks, especially during the COVID-19 pandemic. However, COVID-19 disease models built from wastewater-collected data have been limited by the complexities involved in estimating SARS-CoV-2 fecal shedding rates. In this study, wastewater from six municipalities in Arizona and Florida with distinct demographics were monitored for SARS-CoV-2 RNA between September 2020 and December 2021. Virus concentrations with corresponding clinical case counts were utilized to estimate community-wide fecal shedding rates that encompassed all infected individuals. Analyses suggest that average SARS-CoV-2 RNA fecal shedding rates typically occurred within a consistent range (7.53-9.29 log10 gc/g-feces); and yet, were unique to each community and influenced by population demographics. Age, ethnicity, and socio-economic factors may have influenced shedding rates. Interestingly, populations with median age between 30 and 39 had the greatest fecal shedding rates. Additionally, rates remained relatively constant throughout the pandemic provided conditions related to vaccination and variants were unchanged. Rates significantly increased in some communities when the Delta variant became predominant. Findings in this study suggest that community-specific shedding rates may be appropriate in model development relating wastewater virus concentrations to clinical case counts.

Selected Mechanistic Aspects of Viral Inactivation by Peracetic Acid.

Peracetic acid (PAA) is an alternative to traditional wastewater disinfection as it has a high oxidation potential without producing chlorinated disinfection byproducts. Reports have shown the effectiveness of PAA to reduce waterborne viruses, but the mechanism of inactivation is understudied. This study evaluated PAA consumption by amino acids and nucleotides that are the building blocks of both viral capsids and genomes. Cysteine (>1.7 min-1) and methionine (>1.2 min-1) rapidly consumed PAA, while cystine (1.9 × 10-2 min-1) and tryptophan (1.4 × 10-4 min-1) reactions occurred at a slower rate. All other amino acids and nucleotides did not react significantly (p < 0.05) with PAA during experiments. Also, PAA treatment did not result in significant (p < 0.05) reductions of purified RNA from MS2 bacteriophage and murine norovirus. Data in this study suggest that PAA effectively inactivates viruses by targeting susceptible amino acids on capsid proteins and does not readily damage viral genomes. Knowledge of virus capsid structures and protein compositions can be used to qualitatively predict the relative resistance or susceptibility of virus types to PAA. Capsid structures containing a higher total number of target amino acids may be more susceptible to PAA reactions that damage structural integrity resulting in inactivation.

Enumerating asymptomatic COVID-19 cases and estimating SARS-CoV-2 fecal shedding rates via wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) was utilized to monitor SARS-CoV-2 RNA in sewage collected from manholes specific to individual student dormitories (dorms) at the University of Arizona in the fall semester of 2020, which led to successful identification and reduction of SARS-CoV-2 transmission events. Positive wastewater samples triggered clinical testing of residents within that dorm; thus, SARS-CoV-2 infected individuals were identified regardless of symptom expression. This current study examined clinical testing data to determine the abundance of asymptomatic versus symptomatic cases in these defined communities. Nasal and nasopharyngeal swab samples processed via antigen and PCR tests indicated that 79.2% of SARS-CoV-2 infections were asymptomatic, and only 20.8% of positive cases reported COVID-19 symptoms at the time of testing. Clinical data was paired with corresponding wastewater virus concentrations, which enabled calculation of viral shedding rates in feces per infected person. Mean shedding rates averaged from positive wastewater samples across all dorms were 7.30 ± 0.67 log10 genome copies per gram of feces (gc/g-feces) based on the N1 gene. Quantification of SARS-CoV-2 fecal shedding rates from infected individuals has been the critical missing component necessary for WBE models to measure and predict SARS-CoV-2 infection prevalence in communities. The findings from this study can be utilized to create models that can be used to inform public health prevention and response actions.

Occurrence of SARS-CoV-2 RNA in Six Municipal Wastewater Treatment Plants at the Early Stage of COVID-19 Pandemic in The United States.

In this study, we investigated the occurrence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) RNA in primary influent (n = 42), secondary effluent (n = 24) and tertiary treated effluent (n = 34) collected from six wastewater treatment plants (WWTPs A-F) in Virginia (WWTP A), Florida (WWTPs B, C, and D), and Georgia (WWTPs E and F) in the United States during April-July 2020. Of the 100 wastewater samples analyzed, eight (19%) untreated wastewater samples collected from the primary influents contained SARS-CoV-2 RNA as measured by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. SARS-CoV-2 RNA were detected in influent wastewater samples collected from WWTP A (Virginia), WWTPs E and F (Georgia) and WWTP D (Florida). Secondary and tertiary effluent samples were not positive for SARS-CoV-2 RNA indicating the treatment processes in these WWTPs potentially removed SARS-CoV-2 RNA during the secondary and tertiary treatment processes. However, further studies are needed to understand the log removal values (LRVs) and transmission risks of SARS-CoV-2 RNA through analyzing wastewater samples from a wider range of WWTPs.

COVID-19 containment on a college campus via wastewater-based epidemiology, targeted clinical testing and an intervention.

Wastewater-based epidemiology has potential as an early-warning tool for determining the presence of COVID-19 in a community. The University of Arizona (UArizona) utilized WBE paired with clinical testing as a surveillance tool to monitor the UArizona community for SARS-CoV-2 in near real-time, as students re-entered campus in the fall. Positive detection of virus RNA in wastewater lead to selected clinical testing, identification, and isolation of three infected individuals (one symptomatic and two asymptomatic) that averted potential disease transmission. This case study demonstrated the value of WBE as a tool to efficiently utilize resources for COVID-19 prevention and response. Thus, WBE coupled with targeted clinical testing was further conducted on 13 dorms during the course of the Fall semester (Table 3). In total, 91 wastewater samples resulted in positive detection of SARS-CoV-2 RNA that successfully provided an early-warning for at least a single new reported case of infection (positive clinical test) among the residents living in the dorm. Overall, WBE proved to be an accurate diagnostic for new cases of COVID-19 with an 82.0% positive predictive value and an 88.9% negative predictive value. Increases in positive wastewater samples and clinical tests were noted following holiday-related activities. However, shelter-in-place policies proved to be effective in reducing the number of daily reported positive wastewater and clinical tests. This case study provides evidence for WBE paired with clinical testing and public health interventions to effectively contain potential outbreaks of COVID-19 in defined communities.

First detection of SARS-CoV-2 RNA in wastewater in North America: A study in Louisiana, USA.

We investigated the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater samples in southern Louisiana, USA. Untreated and treated wastewater samples were collected on five occasions over a four-month period from January to April 2020. The wastewater samples were concentrated via ultrafiltration (Method A), and an adsorption-elution method using electronegative membranes (Method B). SARS-CoV-2 RNA was detected in 2 out of 15 wastewater samples using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays (CDC N1 and N2). None of the secondary treated and final effluent samples tested positive for SARS-CoV-2 RNA. To our knowledge, this is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in North America, including the USA. However, concentration methods and RT-qPCR assays need to be refined and validated to increase the sensitivity of SARS-CoV-2 RNA detection in wastewater.

Reduction of erythromycin resistance gene erm(F) and class 1 integron-integrase genes in wastewater by Bardenpho treatment.

Wastewaters routinely contain antibiotic-resistant bacteria (ARB) and genes (ARG) that are removed to a varying degree during wastewater treatment. This study investigated the removal of the erythromycin ribosome methylase class F (erm(F)) and class 1 integron-integrase (intI1) genes at each stage from two water resource recovery facilities in southern Arizona. Although genes were significantly reduced by Bardenpho treatment, erm(F) and intI1 were still observed in ≥ 9 and 7 out of 12 secondary effluent samples. Primary processes via sedimentation or dissolved air flotation, as well as chlorine disinfection, did not significantly impact erm(F) and intI1 concentrations. Therefore, Bardenpho treatment was critical to reduce erm(F) and intI1. Concentrations of erm(F) and intI1 were compared with each other and other markers for anthropogenic pollution. Results from this study support intI1 as one suitable marker to measure erythromycin resistance genes in wastewater, as intI1 was found at higher concentrations, persisted more throughout treatment, and correlated with erm(F) at nearly every treatment stage. PRACTITIONER POINTS: Bardenpho treatment was the key process responsible for the reduction of intI1 and erm(F) genes during wastewater treatment. Primary treatment and chlorine disinfection did not impact erm(F) and intI1 gene concentrations. The intI1 gene is a suitable marker for measuring erm(F) genes in wastewater.

Metagenomic and Resistome Analysis of a Full-Scale Municipal Wastewater Treatment Plant in Singapore Containing Membrane Bioreactors.

Reclaimed water provides a water supply alternative to address problems of scarcity in urbanized cities with high living densities and limited natural water resources. In this study, wastewater metagenomes from 6 stages of a wastewater treatment plant (WWTP) integrating conventional and membrane bioreactor (MBR) treatment were evaluated for diversity of antibiotic resistance genes (ARGs) and bacteria, and relative abundance of class 1 integron integrases (intl1). ARGs confering resistance to 12 classes of antibiotics (ARG types) persisted through the treatment stages, which included genes that confer resistance to aminoglycoside [aadA, aph(6)-I, aph(3')-I, aac(6')-I, aac(6')-II, ant(2″)-I], beta-lactams [class A, class C, class D beta-lactamases (bla OXA)], chloramphenicol (acetyltransferase, exporters, floR, cmIA), fosmidomycin (rosAB), macrolide-lincosamide-streptogramin (macAB, ereA, ermFB), multidrug resistance (subunits of transporters), polymyxin (arnA), quinolone (qnrS), rifamycin (arr), sulfonamide (sul1, sul2), and tetracycline (tetM, tetG, tetE, tet36, tet39, tetR, tet43, tetQ, tetX). Although the ARG subtypes in sludge and MBR effluents reduced in diversity relative to the influent, clinically relevant beta lactamases (i.e., bla KPC, bla OXA) were detected, casting light on other potential point sources of ARG dissemination within the wastewater treatment process. To gain a deeper insight into the types of bacteria that may survive the MBR removal process, genome bins were recovered from metagenomic data of MBR effluents. A total of 101 close to complete draft genomes were assembled and annotated to reveal a variety of bacteria bearing metal resistance genes and ARGs in the MBR effluent. Three bins in particular were affiliated to Mycobacterium smegmatis, Acinetobacter Iwoffii, and Flavobacterium psychrophila, and carried aquired ARGs aac(2')-Ib, bla OXA-278, and tet36 respectively. In terms of indicator organisms, cumulative log removal values (LRV) of Escherichia coli, Enterococci, and P. aeruginosa from influent to conventional treated effluent was lower (0-2.4), compared to MBR effluent (5.3-7.4). We conclude that MBR is an effective treatment method for reducing fecal indicators and ARGs; however, incomplete removal of P. aeruginosa in MBR treated effluents (<8 MPN/100 mL) and the presence of ARGs and intl1 underscores the need to establish if further treatment should be applied prior to reuse.

Assessing the spatial and temporal variability of bacterial communities in two Bardenpho wastewater treatment systems via Illumina MiSeq sequencing.

Next generation sequencing provides new insights into the diversity and ecophysiology of bacteria communities throughout wastewater treatment plants (WWTP), as well as the fate of pathogens in wastewater treatment system. In the present study, we investigated the bacterial communities and human-associated Bacteroidales (HF183) marker in two WWTPs in North America that utilize Bardenpho treatment processes. Although, most pathogens were eliminated during wastewater treatment, some pathogenic bacteria were still observed in final effluents. The HF183 genetic marker demonstrated significant reductions between influent and post-Bardenpho treated samples in each WWTP, which coincided with changes in bacteria relative abundances and community compositions. Consistent with previous studies, the major phyla in wastewater samples were predominantly comprised by Proteobacteria (with Gammaproteobacteria and Alphaproteobacteria among the top two classes), Actinobacteria, Bacteroidetes, and Firmicutes. Dominant genera were often members of Proteobacteria and Firmicutes, including several pathogens of public health concern, such as Pseudomonas, Serratia, Streptococcus, Mycobacterium and Arcobacter. Pearson correlations were calculated to observe the seasonal variation of relative abundances of gene sequences at different levels based on the monthly average temperature. These findings profile how changes in bacterial communities can function as a robust method for monitoring wastewater treatment quality and performance for public and environmental health purposes.

Occurrence of Traditional and Alternative Fecal Indicators in Tropical Urban Environments under Different Land Use Patterns.

This study evaluated the geospatial distribution of fecal indicator bacteria (FIB) (i.e., Escherichia coli, Enterococcus spp.) and the alternative fecal indicator pepper mild mottle virus (PMMoV) in tropical freshwater environments under different land use patterns. Results show that the occurrence and concentration of microbial fecal indicators were higher for urban than for parkland-dominated areas, consistent with land use weightage. Significant positive correlations with traditional FIB indicate that PMMoV is a suitable indicator of fecal contamination in tropical catchments waters (0.549 ≤ rho ≤ 0.612; P < 0.01). PMMoV exhibited a strong significant correlation with land use weightage (rho = 0.728; P < 0.01) compared to traditional FIB (rho = 0.583; P < 0.01). In addition, chemical tracers were also added to evaluate the potential relationships with microbial fecal indicators. The relationships between diverse variables (e.g., environmental parameters, land use coverage, and chemical tracers) and the occurrence of FIB and PMMoV were evaluated. By using stepwise multiple linear regression (MLR), the empirical experimental models substantiate the impact of land use patterns and anthropogenic activities on microbial water quality, and the output results of the empirical models may be able to predict the sources and transportation of human fecal pollution or sewage contamination. In addition, the high correlation between PMMoV data obtained from quantitative real-time PCR (qPCR) and viral metagenomics data supports the possibility of using viral metagenomics to relatively quantify specific microbial indicators for monitoring microbial water quality (0.588 ≤ rho ≤ 0.879; P < 0.05).IMPORTANCE The results of this study may support the hypothesis of using PMMoV as an alternative indicator of human fecal contamination in tropical surface waters from the perspective of land use patterns. The predictive result of the occurrence of human fecal indicators with high accuracy may reflect the source and transportation of human fecal pollution, which are directly related to the risk to human health, and thereafter, steps can be taken to mitigate these risks.

Reduction of Cryptosporidium, Giardia, and Fecal Indicators by Bardenpho Wastewater Treatment.

Increased demand for water reuse and reclamation accentuates the importance for optimal wastewater treatment to limit protozoa in effluents. Two wastewater treatment plants utilizing advanced Bardenpho were investigated over a 12-month period to determine the incidence and reduction of Cryptosporidium, Giardia, Cyclospora, and fecal indicators. Results were compared to facilities that previously operated in the same geographical area. Protozoa (oo)cysts were concentrated using an electronegative filter and subsequently detected by fluorescent microscopy and/or PCR methods. Cryptosporidium and Giardia were frequently detected in raw sewage, but Cyclospora was not detected in any wastewater samples. Facilities with Bardenpho treatment exhibited higher removals of (oo)cysts than facilities utilizing activated sludge or trickling filters. This was likely due to Bardenpho systems having increased solid wasting rates; however, this mechanism cannot be confirmed as sludge samples were not analyzed. Use of dissolved-air-flotation instead of sedimentation tanks did not result in more efficient removal of (oo)cysts. Concentrations of protozoa were compared with each other, Escherichia coli, somatic coliphage, and viruses (pepper mild mottle virus, Aichi virus 1, adenovirus, and polyomaviruses JC and BK). Although significant correlations were rare, somatic coliphage showed the highest potential as an indicator for the abundance of protozoa in wastewaters.

Virus Reduction during Advanced Bardenpho and Conventional Wastewater Treatment Processes.

The present study investigated wastewater treatment for the removal of 11 different virus types (pepper mild mottle virus; Aichi virus; genogroup I, II, and IV noroviruses; enterovirus; sapovirus; group-A rotavirus; adenovirus; and JC and BK polyomaviruses) by two wastewater treatment facilities utilizing advanced Bardenpho technology and compared the results with conventional treatment processes. To our knowledge, this is the first study comparing full-scale treatment processes that all received sewage influent from the same region. The incidence of viruses in wastewater was assessed with respect to absolute abundance, occurrence, and reduction in monthly samples collected throughout a 12 month period in southern Arizona. Samples were concentrated via an electronegative filter method and quantified using TaqMan-based quantitative polymerase chain reaction (qPCR). Results suggest that Plant D, utilizing an advanced Bardenpho process as secondary treatment, effectively reduced pathogenic viruses better than facilities using conventional processes. However, the absence of cell-culture assays did not allow an accurate assessment of infective viruses. On the basis of these data, the Aichi virus is suggested as a conservative viral marker for adequate wastewater treatment, as it most often showed the best correlation coefficients to viral pathogens, was always detected at higher concentrations, and may overestimate the potential virus risk.