Health Risk from the Use of Roof-Harvested Rainwater in Southeast Queensland,Australia, as Potable or Nonpotable Water,Determined Using Quantitative Microbial Risk Assessment

A total of 214 rainwater samples from 82 tanks were collected in urban Southeast Queensland (SEQ) in Australia and analyzed for the presence and numbers of zoonotic bacterial and protozoal pathogens using binary PCR and quantitative PCR (qPCR). Quantitative microbial risk assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to potential pathogens from roof-harvested rainwater used as potable or nonpotable water. Of the 214 samples tested, 10.7%, 9.8%, 5.6%, and 0.4% were positive for the Salmonella invA, Giardia lamblia β-giardin, Legionella pneumophila mip, and Campylobacter jejuni mapA genes, respectively. Cryptosporidium parvum oocyst wall protein (COWP) could not be detected. The estimated numbers of Salmonella, G. lamblia, and L. pneumophila organisms ranged from 6.5 × 10¹ to 3.8 × 10² cells, 0.6 × 10° to 3.6 × 10° cysts, and 6.0 × 10¹ to 1.7 × 10² cells per 1,000 ml of water, respectively. Six risk scenarios were considered for exposure to Salmonella spp., G. lamblia, and L. pneumophila. For Salmonella spp. and G. lamblia, these scenarios were (i) liquid ingestion due to drinking of rainwater on a daily basis, (ii) accidental liquid ingestion due to hosing twice a week, (iii) aerosol ingestion due to showering on a daily basis, and (iv) aerosol ingestion due to hosing twice a week. For L. pneumophila, these scenarios were (i) aerosol inhalation due to showering on a daily basis and (ii) aerosol inhalation due to hosing twice a week. The risk of infection from Salmonella spp., G. lamblia, and L. pneumophila associated with the use of rainwater for showering and garden hosing was calculated to be well below the threshold value of one extra infection per 10,000 persons per year in urban SEQ. However, the risk of infection from ingesting Salmonella spp. and G. lamblia via drinking exceeded this threshold value and indicated that if undisinfected rainwater is ingested by drinking, then the incidences of the gastrointestinal diseases salmonellosis and giardiasis are expected to range from 9.8 × 10° to 5.4 × 10¹ (with a mean of 1.2 × 10¹ from Monte Carlo analysis) and from 1.0 × 10¹ to 6.5 × 10¹ cases (with a mean of 1.6 × 10¹ from Monte Carlo analysis) per 10,000 persons per year, respectively, in urban SEQ. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically examined. Nonetheless, it would seem prudent to disinfect rainwater for use as potable water.Roof-harvested rainwater has received significant attention as a potential alternative source of potable and nonpotable water in regions where water is scarce (37). To encourage the use of roof-harvested rainwater, governmental bodies of many countries, such as Australia, Denmark, Germany, India, and New Zealand, are providing subsidies to residents to encourage the use of rainwater for domestic purposes. The use of rainwater is quite common in Australia, particularly in rural and remote areas, where reticulated mains or town water is not available. Recent water scarcity in several capital cities prompted the use of rainwater as an alternative source. For instance, the Queensland State Government initiated the “Home Water Wise Rebate Scheme,” which provides subsidies to Southeast Queensland (SEQ) residents who use rainwater as nonpotable water for domestic purposes (49). Over 260,000 householders were granted subsidies up to December 2008, when the scheme was concluded.There is a general community feeling that roof-harvested rainwater is safe to drink, and this is partially supported by limited epidemiological evidence (26). Some studies have reported that roof-harvested rainwater quality is generally acceptable for use as potable water (13, 29). In contrast, the presence of potential pathogens, such as Aeromonas spp. Campylobacter spp., Campylobacter jejuni, Salmonella spp., Legionella pneumophila, Giardia spp., Giardia lamblia, and Cryptosporidium spp., in roof-harvested rainwater samples has been reported (2, 9, 34, 45, 47, 48). Such pathogens can cause gastrointestinal illness in humans, with nausea, vomiting, and/or diarrhea occurring within 12 to 72 h (Salmonella enterica serovar Typhimurium) to 9 to 15 days (Giardia lamblia) after ingestion of contaminated water. L. pneumophila can cause the respiratory infection pneumonia, and the fatality rate can be 50% in immunocompromised patients (57).Direct routine monitoring of the microbiological quality of source water for all possible pathogens is not economically, technologically, or practically feasible. Consequently, traditional fecal indicators, such as fecal coliforms, Escherichia coli, and enterococci, have long been used to determine the presence of pathogens. Most studies assess the quality of roof-harvested rainwater based on the numbers of these fecal indicators (13, 30). However, the major limitation in using fecal bacteria as indicators is their poor correlation with the presence of pathogenic microorganisms in water (2, 30). An alternative is the measurement of pathogens using traditional culture-based methods. However, there are several limitations of such methods, including the underestimation of the bacterial number due to the presence of injured or stressed cells (10) and the fact that certain microorganisms in environmental waters can be viable but not culturable (39). Culture-based methods are also generally laborious and costly. Recent advances in molecular techniques such as PCR technology enable rapid, specific, and sensitive detection of many pathogens. Advances in PCR methodology also enable the quantification of potential pathogens in source water that are otherwise difficult and/or laborious to culture using traditional microbiological methods. In view of this, we used binary PCR (presence/absence)- and quantitative PCR (qPCR)-based assays to first detect and then quantify zoonotic pathogens in samples from roof-harvested rainwater in SEQ residential houses.The aims of the research study were 2-fold: (i) to quantify the number and frequency of occurrence of Salmonella, G. lamblia, and L. pneumophila organisms in a range of domestic water tanks in SEQ by using qPCR-based methods and (ii) to apply quantitative microbial risk assessment (QMRA) analysis in order to estimate the risk of infection from exposure to these pathogens found in roof-harvested rainwater. The uniqueness of this study stems from the fact that instead of measuring fecal indicators, the pathogens that are capable of causing illness were quantified and this information was combined with QMRA to assess the human health risk of using roof-harvested rainwater as potable or nonpotable water.     

Reversion and T Cell Escape Mutations Compensate the Fitness Loss of a CD8+ T Cell Escape Mutant in Their Cognate Transmitted/Founder Virus

Immune escape mutations that revert back to the consensus sequence frequently occur in newly HIV-1-infected individuals and have been thought to render the viruses more fit. However, their impact on viral fitness and their interaction with other immune escape mutations have not been evaluated in the background of their cognate transmitted/founder (T/F) viral genomes. To precisely determine the role of reversion mutations, we introduced reversion mutations alone or together with CD8⁺ T cell escape mutations in their unmodified cognate T/F viral genome and determined their impact on viral fitness in primary CD4⁺ T cells. Two reversion mutations, V247I and I64T, were identified in Gag and Tat, respectively, but neither had measurable effect on the fitness of their cognate T/F virus. The V247I and G248A mutations that were detected before and concurrently with the potent T cell escape mutation T242N, respectively, were selected by early T cell responses. The V247I or the G248A mutation alone partially restored the fitness loss caused by the T242N mutation. Together they could fully restore the fitness of the T242N mutant to the T/F level. These results demonstrate that the fitness loss caused by a T cell escape mutation could be compensated by preexisting or concurrent reversion and other T cell escape mutations. Our findings indicate that the overall viral fitness is modulated by the complex interplay among T cell escape, compensatory and reversion mutations to maintain the balance between immune escape and viral replication capacity.     

Quantitative PCR assay of sewage-associated Bacteroides markers to assess sewage pollution in an urban lake in Dhaka, Bangladesh

This paper aimed to assess the magnitude of sewage pollution in an urban lake in Dhaka, Bangladesh, by using quantitative PCR of sewage-associated Bacteroides HF183 markers. PCR was also used for the quantitative detection of ruminant wastewater-associated CF128 markers along with the enumeration of traditional fecal indicator bacteria, namely enterococci. The number of enterococci in lake water samples ranged from 1.1 × 10? to 1.9 × 10? colony-forming units/100 mL water. From the 20 water samples tested, 14 (70%) and 7 (35%) were PCR positive for HF183 and CF128 markers, respectively. The numbers of HF183 and CF128 markers in lake water samples were 3.9 × 10? to 6.3 × 10? and 9.3 × 103 to 6.3 × 10? genomic units/100 mL water, respectively. The high numbers of enterococci and HF183 markers are indicative of sewage pollution and potential health risks to those who use the lake water for nonpotable purposes such as bathing and washing clothes. This is the first study that investigated the presence of microbial source tracking markers in Dhaka, Bangladesh, where diarrhoeal disease is one of the major causes of childhood mortality. The molecular assay used in this study can provide valuable information on the extent of sewage pollution, thus facilitating the development of robust strategies to minimize potential health risks.     

Broad TCR usage in functional HIV-1-specific CD8+ T cell expansions driven by vaccination during highly active antiretroviral therapy

During chronic HIV-1 infection, continuing viral replication is associated with impaired proliferative capacity of virus-specific CD8+ T cells and with the expansion and persistence of oligoclonal T cell populations. TCR usage may significantly influence CD8+ T cell-mediated control of AIDS viruses; however, the potential to modulate the repertoire of functional virus-specific T cells by immunotherapy has not been explored. To investigate this, we analyzed the TCR Vbeta usage of CD8+ T cells populations which were expanded following vaccination with modified vaccinia virus Ankara expressing a HIV-1 gag/multiepitope immunogen (MVA.HIVA) in HIV-1-infected patients receiving highly active antiretroviral therapy. Vaccinations induced the re-expansion of HIV-1-specific CD8+ T cells and these showed broad TCR Vbeta usage which was maintained for at least 1 year in some individuals. By contrast, virus-specific CD8+ T cell populations in the same donors which failed to expand after vaccination and in unvaccinated controls were oligoclonal. Simultaneously, we observed that CD8+ T cells recognizing vaccine-derived HIV-1 epitopes displayed enhanced capacity to proliferate and to inhibit HIV-1 replication in vitro, following MVA.HIVA immunizations. Taken together, these data indicate that an attenuated viral-vectored vaccine can modulate adaptive CD8+ T cell responses to HIV-1 and improve their antiviral functional capacity. The potential therapeutic benefit of this vaccination approach warrants further investigation.     

Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes

A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8(+) T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2x MVA.HIVA) (n=8) or two doses of placebo (2x placebo) (n=4). The second group received 2x pTHr.HIVA followed by one dose of MVA.HIVA (n=8) or 3x placebo (n=4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-gamma) ELISPOT (group mean, 210 spot-forming cells/10(6) cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2x MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-gamma ELISPOT assay, positive responses mainly mediated by CD4(+) T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2x MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4(+) T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8(+) T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1 beta. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.     

Sourcing faecal pollution from onsite wastewater treatment systems in surface waters using antibiotic resistance analysis

AIMS: To identify the sources of faecal contamination in investigated surface waters and to determine the significance of onsite wastewater treatment systems (OWTS) as a major contributor to faecal contamination. METHODS AND RESULTS: Antibiotic resistance patterns (ARP) were established for a library of 717 known Escherichia coli source isolates obtained from human, domesticated animals, livestock and wild sources. Eight commonly used antibiotics, including amoxicillin, cephalothin, erythromycin, gentamicin, ofloxacin, chlortetracycline, tetracycline and moxalactam, at four different concentrations were used to obtain ARPs for E. coli isolates. Discriminant analysis (DA) was used to differentiate between the ARP of sources isolates. The developed ARP library was found to be adequate for discriminating human from nonhuman isolates, and was used to classify 256 enumerated E. coli isolates collected from monitored surface water locations. CONCLUSIONS: The resulting ARP DA indicated that a majority of the faecal contamination in more rural areas was nonhuman; however, the percentage of human isolates increased significantly in urbanized areas using OWTS for wastewater treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study signifies the feasibility of using ARP for source tracking faecal contamination in surface waters, and linking faecal contamination to OWTS. The information will enable regulatory authorities to implement appropriate management practices to reduce the contamination of water resources caused by high densities and failing OWTS.     

Reappraisal of the relationship between the HIV-1-protective single-nucleotide polymorphism 35 kilobases upstream of the HLA-C gene and surface HLA-C expression

Previous studies have found an association between a single-nucleotide polymorphism 35 kb upstream of the HLA-C locus (-35 SNP), HLA-C expression, and HIV-1 set point viral loads. We show that the difference in HLA-C expression across -35 SNP genotypes can be attributed primarily to the very low expression of a single allelic product, HLA-Cw7, which is a common HLA type. We suggest that association of the -35 SNP and HIV-1 load manifests as a result of linkage disequilibrium of this polymorphism with both favorable and unfavorable HLA-C and -B alleles.     

Fitness costs and diversity of the cytotoxic T lymphocyte (CTL) response determine the rate of CTL escape during acute and chronic phases of HIV infection

HIV-1 often evades cytotoxic T cell (CTL) responses by generating variants that are not recognized by CTLs. We used single-genome amplification and sequencing of complete HIV genomes to identify longitudinal changes in the transmitted/founder virus from the establishment of infection to the viral set point at 1 year after the infection. We found that the rate of viral escape from CTL responses in a given patient decreases dramatically from acute infection to the viral set point. Using a novel mathematical model that tracks the dynamics of viral escape at multiple epitopes, we show that a number of factors could potentially contribute to a slower escape in the chronic phase of infection, such as a decreased magnitude of epitope-specific CTL responses, an increased fitness cost of escape mutations, or an increased diversity of the CTL response. In the model, an increase in the number of epitope-specific CTL responses can reduce the rate of viral escape from a given epitope-specific CTL response, particularly if CD8+ T cells compete for killing of infected cells or control virus replication nonlytically. Our mathematical framework of viral escape from multiple CTL responses can be used to predict the breadth and magnitude of HIV-specific CTL responses that need to be induced by vaccination to reduce (or even prevent) viral escape following HIV infection.