Quantification and Stability of Human Adenoviruses and Polyomavirus JCPyV in Wastewater Matrices

Human adenoviruses (HAdV) and human polyomavirus JCPyV have been previously proposed as indicators of fecal viral contamination in the environment. Different wastewater matrices have been analyzed by applying real-time quantitative PCR procedures for the presence, quantity, and stability of a wide diversity of excreted HAdV and JCPyV. High quantities of HAdV and JCPyV were detected in sewage, effluent wastewater, sludge, and biosolid samples. Both viruses showed high stability in urban sewage. These results confirm the suitability of both viruses as indicators of human fecal viral pollution.     

Quantification and stability of human adenoviruses and polyomavirus JCPyV in wastewater matrices

Human adenoviruses (HAdV) and human polyomavirus JCPyV have been previously proposed as indicators of fecal viral contamination in the environment. Different wastewater matrices have been analyzed by applying real-time quantitative PCR procedures for the presence, quantity, and stability of a wide diversity of excreted HAdV and JCPyV. High quantities of HAdV and JCPyV were detected in sewage, effluent wastewater, sludge, and biosolid samples. Both viruses showed high stability in urban sewage. These results confirm the suitability of both viruses as indicators of human fecal viral pollution.     

Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water

In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways.     

Relevance of bacteroidales and F-specific RNA bacteriophages for efficient fecal contamination tracking at the level of a catchment in France

The relevance of three host-associated Bacteroidales markers (HF183, Rum2Bac, and Pig2Bac) and four F-specific RNA bacteriophage genogroups (FRNAPH I to IV) as microbial source tracking markers was assessed at the level of a catchment (Daoulas, France). They were monitored together with fecal indicators (Escherichia coli and enterococci) and chemophysical parameters (rainfall, temperature, salinity, pH, and turbidity) by monthly sampling over 2 years (n = 240 water samples) and one specific sampling following an accidental pig manure spillage (n = 5 samples). During the 2-year regular monitoring, levels of E. coli, enterococci, total F-specific RNA bacteriophages, and the general Bacteroidales marker AllBac were strongly correlated with one another and with Rum2Bac (r = 0.37 to 0.50, P < 0.0001). Their correlations with HF183 and FRNAPH I and II were lower (r = 0.21 to 0.29, P < 0.001 to P < 0.0001), and HF183 and enterococci were associated rather than correlated (Fisher's exact test, P < 0.01). Rum2Bac and HF183 enabled 73% of water samples that had ≥ 2.7 log(10) most probably number (MPN) of E. coli/100 ml to be classified. FRNAPH I and II enabled 33% of samples at this contamination level to be classified. FRNAPH I and II complemented the water sample classification obtained with the two Bacteroidales markers by an additional 8%. Pig2Bac and FRNAPH III and IV were observed in a small number of samples (n = 0 to 4 of 245). The present study validates Rum2Bac and HF183 as relevant tools to trace fecal contamination originating from ruminant or human waste, respectively, at the level of a whole catchment.     

Clustering of water bodies in unpolluted and polluted environments based on Escherichia coli phylogroup abundance using a simple interaction database

Different types of water bodies, including lakes, streams, and coastal marine waters, are often susceptible to fecal contamination from a range of point and nonpoint sources, and have been evaluated using fecal indicator microorganisms. The most commonly used fecal indicator is Escherichia coli, but traditional cultivation methods do not allow discrimination of the source of pollution. The use of triplex PCR offers an approach that is fast and inexpensive, and here enabled the identification of phylogroups. The phylogenetic distribution of E. coli subgroups isolated from water samples revealed higher frequencies of subgroups A₁ and B2₃ in rivers impacted by human pollution sources, while subgroups D₁ and D₂ were associated with pristine sites, and subgroup B1 with domesticated animal sources, suggesting their use as a first screening for pollution source identification. A simple classification is also proposed based on phylogenetic subgroup distribution using the w-clique metric, enabling differentiation of polluted and unpolluted sites.     

Identification of Human and Animal Adenoviruses and Polyomaviruses for Determination of Sources of Fecal Contamination in the Environment

The Adenoviridae and Polyomaviridae families comprise a wide diversity of viruses which may be excreted for long periods in feces or urine. In this study, a preliminary analysis of the prevalence in the environment and the potential usefulness as source-tracking tools of human and animal adenoviruses and polyomaviruses has been developed. Molecular assays based on PCR specifically targeting human adenoviruses (HAdV), porcine adenoviruses (PAdV), bovine adenoviruses (BAdV), and bovine polyomaviruses (BPyV) were applied to environmental samples including urban sewage, slaughterhouse, and river water samples. PAdV and BPyV were detected in a very high percentage of samples potentially affected by either porcine or bovine fecal contamination, respectively. However, BAdV were detected in only one sample, showing a lower prevalence than BPyV in the wastewater samples analyzed. The 22 slaughterhouse samples with fecal contamination of animal origin showed negative results for the presence of HAdV. The river water samples analyzed were positive for the presence of both human and animal adenoviruses and polyomaviruses, indicating the existence of diverse sources of contamination. The identities of the viruses detected were confirmed by analyses of the amplified sequences. All BPyV isolates showed a 97% similarity in nucleotide sequences. This is the first time that PAdV5, BAdV6, and BPyV have been reported to occur in environmental samples. Human and porcine adenoviruses and human and bovine polyomaviruses are proposed as tools for evaluating the presence of viral contamination and for tracking the origin of fecal/urine contamination in environmental samples.     

Identification of human and animal adenoviruses and polyomaviruses for determination of sources of fecal contamination in the environment

The Adenoviridae and Polyomaviridae families comprise a wide diversity of viruses which may be excreted for long periods in feces or urine. In this study, a preliminary analysis of the prevalence in the environment and the potential usefulness as source-tracking tools of human and animal adenoviruses and polyomaviruses has been developed. Molecular assays based on PCR specifically targeting human adenoviruses (HAdV), porcine adenoviruses (PAdV), bovine adenoviruses (BAdV), and bovine polyomaviruses (BPyV) were applied to environmental samples including urban sewage, slaughterhouse, and river water samples. PAdV and BPyV were detected in a very high percentage of samples potentially affected by either porcine or bovine fecal contamination, respectively. However, BAdV were detected in only one sample, showing a lower prevalence than BPyV in the wastewater samples analyzed. The 22 slaughterhouse samples with fecal contamination of animal origin showed negative results for the presence of HAdV. The river water samples analyzed were positive for the presence of both human and animal adenoviruses and polyomaviruses, indicating the existence of diverse sources of contamination. The identities of the viruses detected were confirmed by analyses of the amplified sequences. All BPyV isolates showed a 97% similarity in nucleotide sequences. This is the first time that PAdV5, BAdV6, and BPyV have been reported to occur in environmental samples. Human and porcine adenoviruses and human and bovine polyomaviruses are proposed as tools for evaluating the presence of viral contamination and for tracking the origin of fecal/urine contamination in environmental samples.     

Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution.

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is a ubiquitous bacterium in the intestines of warm-blooded animals and is used as an indicator of fecal pollution. However, its presence does not specifically differentiate sources of pollution. A total of 238 E. coli isolates from human sources (HS) and nonhuman sources (NHS) were collected from the Apalachicola National Estuarine Research Reserve, from associated sewage treatment plants, and directly from animals and tested for ribotype (RT) profile. HS and NHS isolates showed 41 and 61 RT profiles, respectively. At a similarity index of ca. 50%, HS and NHS isolates demonstrated four clusters, with the majority of HS and NHS isolates located in clusters C and D; isolates obtained directly from human and animal feces also could be grouped within these clusters. Discriminant analysis (DA) of RT profiles showed that 97% of the NHS isolates and 100% of the animal fecal isolates were correctly classified. The average rate of correct classification for HS and NHS isolates was 82%. We conclude that DA of RT profiles may be a useful method for identifying HS and NHS fecal pollution and may potentially facilitate management practices.     

Diversity of bacteroides fragilis strains in their capacity to recover phages from human and animal wastes and from fecally polluted wastewater

Great differences in capability to detect bacteriophages from urban sewage of the area of Barcelona existed among 115 strains of Bacteroides fragilis. The capability of six of the strains to detect phages in a variety of feces and wastewater was studied. Strains HSP40 and RYC4023 detected similar numbers of phages in urban sewage and did not detect phages in animal feces. The other four strains detected phages in the feces of different animal species and in wastewater of both human and animal origin. Strain RYC2056 recovered consistently higher counts than the other strains and also detected counts ranging from 10(1) to approximately 10(3) phages per ml in urban sewage from different geographical areas. This strain detected bacteriophages in animal feces even though their relative concentration with respect to the other fecal indicators was significantly lower in wastewater polluted with animal feces than in urban sewage.     

Fecal Pollution in Coastal Marine Sediments from a Semi-Enclosed Deep Embayment Subjected to Anthropogenic Activities: An Issue to Be Considered in Environmental Quality Management Frameworks Development

Sewage discharge is a major source of pollution in marine environments. Urban wastewaters can directly enter marine environments carrying pathogen organisms, organic loads, and nutrients. Because marine sediments can act as the ultimate fate of a wide range of pollutants, environmental quality assessment in this compartment can help to identify pollution problems in coastal areas. In the present study, characterization of surficial marine sediments allowed assessment of fecal pollution in a semi-enclosed deep embayment that is subjected to anthropogenic activities. Physicochemical parameters and fecal indicators presented a great spatial heterogeneity. Fecal coliform and Clostridium perfringens showed accumulation in an extensive area, not only in proximity to sewage discharge points, but also in sediments at 100 meters depth. Results included herein demonstrated that, in coastal areas, urban wastewater discharge can affect the whole ecosystem through accumulation of fecal matter in bottom sediments. Application of multivariate techniques provided useful information with applicability for management of coastal areas in such complex systems. Environmental implications of wastewater discharge in coastal areas indicate the need to implement and include sediment quality control strategies in legislative frameworks.     

Occurrence of Rhodococcus coprophilus and associated actinomycetes in feces, sewage, and freshwater.

Freshwater, sewage, and fecal samples from various sources were examined for Rhodococcus coprophilus, associated actinomycetes, Escherichia coli, and fecal streptococci. Rhodococcus coprophilus was isolated consistently from feces of farm animals, poultry reared in proximity to farm animals, freshwater, and wastewater polluted with animal fecal wastes. It was not isolated from samples of human feces. The ratio of R. coprophilus total actinomycetes was higher in feces from cattle, sheep, ducks, and geese than in specimens from pigs, horses, and fowl. In samples from two freshwater streams polluted by fecal material from farm animals, the ratios of R. copropilus to total actinomycetes were similar to those found in fecal specimens from cattle and sheep. Ratios of fecal coliform to fecal streptococci could not distinguish between fresh human and animal fecal samples and, furthermore, were not reflected in the stream waters polluted by animal fecal material. R. coprophilus has potential in water and dairy bacteriology as a specific indicator organism of fecal pollution due to farm animal wastes.     

New molecular quantitative PCR assay for detection of host-specific Bifidobacteriaceae suitable for microbial source tracking

Bifidobacterium spp. belong to the commensal intestinal microbiota of warm-blooded animals. Some strains of Bifidobacterium show host specificity and have thus been proposed as host-specific targets to determine the origin of fecal pollution. Most strains have been used in microbial-source-tracking (MST) studies based on culture-dependent methods. Although some of these approaches have proved very useful, the low prevalence of culturable Bifidobacterium strains in the environment means that molecular culture-independent procedures could provide practical applications for MST. Reported here is a set of common primers and four Bifidobacterium sp. host-associated (human, cattle, pig, and poultry) probes for quantitative-PCR (qPCR) assessment of fecal source tracking. This set was tested using 25 water samples of diverse origin: urban sewage samples, wastewater from four abattoirs (porcine, bovine, and poultry), and water from a river with a low pollution load. The selected sequences showed a high degree of host specificity. There were no cross-reactions between the qPCR assays specific for each origin and samples from different fecal origins. On the basis of the findings, it was concluded that the host-specific qPCRs are sufficiently robust to be applied in environmental MST studies.     

Diversity of Bacteroides fragilis Strains in Their Capacity To Recover Phages from Human and Animal Wastes and from Fecally Polluted Wastewater

Great differences in capability to detect bacteriophages from urban sewage of the area of Barcelona existed among 115 strains of Bacteroides fragilis. The capability of six of the strains to detect phages in a variety of feces and wastewater was studied. Strains HSP40 and RYC4023 detected similar numbers of phages in urban sewage and did not detect phages in animal feces. The other four strains detected phages in the feces of different animal species and in wastewater of both human and animal origin. Strain RYC2056 recovered consistently higher counts than the other strains and also detected counts ranging from 10¹ to approximately 10³ phages per ml in urban sewage from different geographical areas. This strain detected bacteriophages in animal feces even though their relative concentration with respect to the other fecal indicators was significantly lower in wastewater polluted with animal feces than in urban sewage.     

Microbial Source Tracking for Identification of Fecal Pollution

Fecal pollution is a serious environmental problem that affects many coastal and inland waters worldwide. Both human and animal fecal pollution impose risks to human health from exposure to pathogenic bacteria, viruses, and protozoa. To assist authorities with the implementation of the changes suggested by more restricted legislation concering water quality in Europe, methods are needed which can identify the sources of fecal pollution. Management of fecal contamination of water would be improved if the origin of the fecal pollution could be correctly identified since remediation efforts could then be allocated in a more effective manner. The concept that the origin of fecal pollution can be traced has been termed microbial source tracking. In microbial source tracking (MST) endogenous markers of fecal sources are used for identification of the fecal pollution in aquatic environments. Chemical MST-methods can be used to trace mainly sewage pollution, but the used chemical targets have no direct relationship with pathogenic bacteria. This is not the case in microbial MST-methods where source-specific bacteria or viruses are cultured to identify fecal pollution sources. However, sometimes these microbial targets can be present in too low numbers to be detected. This is circumvented by using molecular assays for host-specific marker detection. Phenotypic and genotypic library-based methods can be used to discriminate among different fecal sources. However, the isolation step makes this procedure very labour-intensive, and issues as temporal and geographical variability remain unresolved. The underlying assumptions will be discussed and the methods mostly used in microbial source tracking will be described in more detail.     

HIV-1 Superinfection in the Antiretroviral Therapy Era: Are Seroconcordant Sexual Partners at Risk?

Background

Acquisition of more than one strain of human immunodeficiency virus type 1 (HIV-1) has been reported to occur both during and after primary infection, but the risks and repercussions of dual and superinfection are incompletely understood. In this study, we evaluated a longitudinal cohort of chronically HIV-infected men who were sexual partners to determine if individuals acquired their partners'' viral strains.

Methodology

Our cohort of HIV-positive men consisted of 8 couples that identified themselves as long-term sexual partners. Viral sequences were isolated from each subject and analyzed using phylogenetic methods. In addition, strain-specific PCR allowed us to search for partners'' viruses present at low levels. Finally, we used computational algorithms to evaluate for recombination between partners'' viral strains.

Principal Findings/Conclusions

All couples had at least one factor associated with increased risk for acquisition of new HIV strains during the study, including detectable plasma viral load, sexually transmitted infections, and unprotected sex. One subject was dually HIV-1 infected, but neither strain corresponded to that of his partner. Three couples'' sequences formed monophyletic clusters at the entry visit, with phylogenetic analysis suggesting that one member of the couple had acquired an HIV strain from his identified partner or that both had acquired it from the same source outside their partnership. The 5 remaining couples initially displayed no evidence of dual infection, using phylogenetic analysis and strain-specific PCR. However, in 1 of these couples, further analysis revealed recombinant viral strains with segments of viral genomes in one subject that may have derived from the enrolled partner. Thus, chronically HIV-1 infected individuals may become superinfected with additional HIV strains from their seroconcordant sexual partners. In some cases, HIV-1 superinfection may become apparent when recombinant viral strains are detected.     

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.     

Reliability at the Lower Limits of HIV-1 RNA Quantification in Clinical Samples: A Comparison of RT-PCR versus bDNA Assays

Introduction

To explore whether an assay change was responsible for an increasing proportion of patients with undetectable HIV viral loads at our urban HIV clinic, we selected highly stable patients, examining their viral loads before and after changing assays. We compared the proportion with detectable viremia during RT-PCR vs. bDNA periods.

Methodology/Principal Findings

We selected patients with ≥1 viral loads assessed during both RT-PCR and bDNA periods. We included patients with stable CD4 counts, excluding patients with viral loads ≥1,000 copies/ml or any significant changes in therapy. Out of 4500 clinic patients, 419 patients (1588 viral loads) were included. 39% of viral loads were reported as detectable by RT-PCR vs. 5% reported as detectable by bDNA. The mean coefficient of variation was higher before vs. after assay change. We found an odds'' ratio of 16.7 for having a viral load >75 copies/ml during the RT-PCR vs. bDNA periods.

Discussion

These data support previous reports, suggesting that bDNA may more reliably discriminate between viral suppression and low level viremia in stable patients on therapy. Low-level viremia, noted more with RT-PCR, may promote unneeded testing, while differences in viral load reliability may impact antiretroviral trial and quality assurance endpoints. Commonly used plasma separator tubes may differentially affect RT-PCR and bDNA results.     

Correlation between bacterial indicators and bacteriophages in sewage and sludge

The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA-specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated wastewater and sludge is presented in this study. Raw and treated wastewater and sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analysed for total coliforms, Escherichia coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. SOMCPH may be used as additional indicators, because of their high densities and resistance to various treatment steps.