h Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters. N umerous host-associated indicators are used to identify human fecal pollution in ambient surface waters, with many relying on molecular methods such as quantitative real-time PCR (qPCR). The most widely used methods target the HF183 16S rRNA gene cluster of members of the genus Bacteroides initially identified in human fecal samples collected from the United States Pacific Northwest (1) and targeted by PCR to distinguish humanassociated contamination (2). Over the past decade, researchers have developed (2-9) and implemented (10-15) many PCR-based methods targeting the HF183 cluster worldwide.In most HF183 PCR applications, the forward primer targets the Bacteroides HF183 cluster, reported to include Bacteroides dorei (3). The reverse primer typically hybridizes to a wider diversity of Bacteroidales so as not to further restrict the range of targeted bacteria. Researchers have employed this strategy for endpoint PCR applications (2), as well as a variety of quantitative real-time PCR chemistries, such as the SYBR green (8, 16) and TaqMan (4, 5, 9, 17) chemistries.In recent performance evaluation studies, PCR-based methods targeting the HF183 cluster, in particular, the TaqMan HF183/ BFDrev assay (3), consistently outperformed other tested approaches in terms of specificity and sensitivity (18-21). TaqMan chemistry utilizes an internal oligonucleotide probe, greatly reducing the chance of false-positive results due to the accumulation of unintended amplification by-products (e.g., primer dimerization [PD] molecules). The TaqMan HF183/BFDrev qPCR assay was recently tested in a five-laboratory repeatability study and shown to be highly reproducible across laboratories when key components of the protocol were standardized (22). However, the ...

Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses.

Available filtration methods to concentrate waterborne viruses are either too costly for studies requiring large numbers of samples, limited to small sample volumes, or not very portable for routine field applications. Sodocalcic glass wool filtration is a cost-effective and easy-to-use method to retain viruses, but its efficiency and reliability are not adequately understood. This study evaluated glass wool filter performance to concentrate the four viruses on the U.S. Environmental Protection Agency contaminant candidate list, i.e., coxsackievirus, echovirus, norovirus, and adenovirus, as well as poliovirus. Total virus numbers recovered were measured by quantitative reverse transcription-PCR (qRT-PCR); infectious polioviruses were quantified by integrated cell culture (ICC)-qRT-PCR. Recovery efficiencies averaged 70% for poliovirus, 14% for coxsackievirus B5, 19% for echovirus 18, 21% for adenovirus 41, and 29% for norovirus. Virus strain and water matrix affected recovery, with significant interaction between the two variables. Optimal recovery was obtained at pH 6.5. No evidence was found that water volume, filtration rate, and number of viruses seeded influenced recovery. The method was successful in detecting indigenous viruses in municipal wells in Wisconsin. Long-term continuous filtration retained viruses sufficiently for their detection for up to 16 days after seeding for qRT-PCR and up to 30 days for ICC-qRT-PCR. Glass wool filtration is suitable for large-volume samples (1,000 liters) collected at high filtration rates (4 liters min ؊1 ), and its low cost makes it advantageous for studies requiring large numbers of samples.Waterborne viruses are an important cause of disease, being responsible for 14% of outbreaks (9 of 64 cases) and 38% of illnesses (1,153 of 3,008 cases) associated with drinking water in the United States from 1999 to 2002 (21, 49). During the same period, noroviruses were responsible for 6% (8 of 66 cases) of outbreaks and 17% (348 of 2,093 cases) of illnesses associated with recreational water. If waterborne illnesses of unknown etiology during the period 1999-2002 are included in the above statistics, as these are believed to be of viral origin, up to 56% and 28% of illness cases associated with drinking water and recreational water, respectively, may be attributed to viruses.To detect and quantify waterborne viruses from environmental samples, the first step in the protocol usually requires concentration from a large water volume. Several concentration methods have been developed and applied successfully in the past two decades (see reviews by Wyn-Jones and Sellwood [48] and Grabow [13]). These include adsorption onto (and subsequent elution from) electropositive cartridges and membranes (3,25,27,29,33,35), gauze pads and glass powder (2, 9, 34), electronegative membranes, and microporous materials (1,8,12,16,20,27) and concentration by ultrafiltration (15, 17, 36, 37) and ultracentrifugation (26). Adsorption onto electropositive cartridges, for example, the CUNO 1-MDS Viroso...

Background: Groundwater supplies for drinking water are frequently contaminated with low levels of human enteric virus genomes, yet evidence for waterborne disease transmission is lacking.Objectives: We related quantitative polymerase chain reaction (qPCR)–measured enteric viruses in the tap water of 14 Wisconsin communities supplied by nondisinfected groundwater to acute gastrointestinal illness (AGI) incidence.Methods: AGI incidence was estimated from health diaries completed weekly by households within each study community during four 12-week periods. Water samples were collected monthly from five to eight households per community. Viruses were measured by qPCR, and infectivity assessed by cell culture. AGI incidence was related to virus measures using Poisson regression with random effects.Results: Communities and time periods with the highest virus measures had correspondingly high AGI incidence. This association was particularly strong for norovirus genogroup I (NoV-GI) and between adult AGI and enteroviruses when echovirus serotypes predominated. At mean concentrations of 1 and 0.8 genomic copies/L of NoV-GI and enteroviruses, respectively, the AGI incidence rate ratios (i.e., relative risk) increased by 30%. Adenoviruses were common, but tap-water concentrations were low and not positively associated with AGI. The estimated fraction of AGI attributable to tap-water–borne viruses was between 6% and 22%, depending on the virus exposure–AGI incidence model selected, and could have been as high as 63% among children < 5 years of age during the period when NoV-GI was abundant in drinking water.Conclusions: The majority of groundwater-source public water systems in the United States produce water without disinfection, and our findings suggest that populations served by such systems may be exposed to waterborne viruses and consequent health risks.

A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.