Susan K. Spencer

Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin

ABSTRACT 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.

Concentration of enteroviruses, adenoviruses, and noroviruses from drinking water by use of glass wool filters

ABSTRACT 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.

Viruses in Nondisinfected Drinking Water from Municipal Wells and Community Incidence of Acute Gastrointestinal Illness

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.

Source and transport of human enteric viruses in deep municipal water supply wells.

Until recently, few water utilities or researchers were aware of possible virus presence in deep aquifers and wells. During 2008 and 2009 we collected a time series of virus samples from six deep municipal water-supply wells. The wells range in depth from approximately 220 to 300 m and draw water from a sandstone aquifer. Three of these wells draw water from beneath a regional aquitard, and three draw water from both above and below the aquitard. We also sampled a local lake and untreated sewage as potential virus sources. Viruses were detected up to 61% of the time in each well sampled, and many groundwater samples were positive for virus infectivity. Lake samples contained viruses over 75% of the time. Virus concentrations and serotypes observed varied markedly with time in all samples. Sewage samples were all extremely high in virus concentration. Virus serotypes detected in sewage and groundwater were temporally correlated, suggesting very rapid virus transport, on the order of weeks, from the source(s) to wells. Adenovirus and enterovirus levels in the wells were associated with precipitation events. The most likely source of the viruses in the wells was leakage of untreated sewage from sanitary sewer pipes.

Use of Streptogramin Growth Promoters in Poultry and Isolation of Streptogramin-Resistant Enterococcus faecium from Humans

BACKGROUND Virginiamycin use in poultry selects for Enterococcus faecium with cross-resistance to quinupristin-dalfopristin, a drug for vancomycin-resistant E. faecium in humans. We conducted an epidemiologic study of poultry exposures as risk factors for human carriage of quinupristin-dalfopristin-resistant E. faecium. METHODS Rectal or fecal samples for E. faecium testing were obtained from 567 newly admitted hospital patients and 100 healthy vegetarians. Participants were interviewed regarding poultry exposure. Retail poultry washes (160 conventional and 26 antibiotic free) were also tested for the presence of E. faecium. Constitutive and inducible quinupristin-dalfopristin resistance were assessed in E. faecium isolates, and resistance genes were identified by polymerase chain reaction. RESULTS E. faecium was isolated from 105 patients, 65 vegetarians, and 77 conventional and 23 antibiotic-free poultry washes. Constitutive quinupristin-dalfopristin resistance was absent in human E. faecium, but 56% of conventional poultry isolates were quinupristin-dalfopristin resistant. Inducible quinupristin-dalfopristin resistance was more common in samples from patients than in those from vegetarians and in washes of conventional than antibiotic-free poultry. Higher poultry consumption was associated with inducible quinupristin-dalfopristin resistance. vatE was present in 38% of E. faecium isolates from patients and none from vegetarians. Touching raw poultry was associated with the presence of vatE. CONCLUSIONS Poultry exposure is associated with a quinupristin-dalfopristin resistance gene and inducible quinupristin-dalfopristin resistance in human fecal E. faecium. The continued use of virginiamycin may increase the potential for streptogramin-resistant E. faecium infection in humans.

Virus contamination from operation and maintenance events in small drinking water distribution systems

We tested the association of common events in drinking water distribution systems with contamination of household tap water with human enteric viruses. Viruses were enumerated by qPCR in the tap water of 14 municipal systems that use non-disinfected groundwater. Ultraviolet disinfection was installed at all active wellheads to reduce virus contributions from groundwater to the distribution systems. As no residual disinfectant was added to the water, any increase in virus levels measured downstream at household taps would be indicative of distribution system intrusions. Utility operators reported events through written questionnaires. Virus outcome measures were related to distribution system events using binomial and gamma regression. Virus concentrations were elevated in the wells, reduced or eliminated by ultraviolet disinfection, and elevated again in distribution systems, showing that viruses were, indeed, directly entering the systems. Pipe installation was significantly associated with higher virus levels, whereas hydrant flushing was significantly associated with lower virus levels. Weak positive associations were observed for water tower maintenance, valve exercising, and cutting open a water main. Coliform bacteria detections from routine monitoring were not associated with viruses. Understanding when distribution systems are most vulnerable to virus contamination, and taking precautionary measures, will ensure delivery of safe drinking water.

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.

Multi-drug resistance in chronic lymphocytic leukemia

We evaluated 45 chronic lymphocyte leukemia (CLL) patients for the presence of multi-drug resistance (MDR) by the ex vivo techniques: 1) a functional assay utilizing doxorubicin (dox) retention with modulation; 2) a cytotoxicity assay (MTT) with modulation; 3) and four monoclonal antibodies. Ex vivo tests were correlated with disease stage and prior treatment, and were repeated as patients became resistant to alkylating agents, fludarabine and VAD chemotherapy (infusion of vincristine, dox, and oral dexamethasone). The majority of patients (64.4%) were in early stage and were untreated (62.2%). P-glycoprotein (p-gp 170) was detected most frequently by the monoclonal antibody MRK-16 (48%) and by functional modulation of dox retention by PSC-833 (40.6%) and by functional modulation of the MTT assay with vincristine (0.29) and dox (0.39) with PSC-833 at 1.0 microg/mL. Functional modulation of dox retention with PSC-833 was significantly associated with stage, but not with either the MTT assay or any of the monoclonal antibodies. None of the tests correlated with prior chlorambucil treatment. Correlation of dox retention with the monoclonal antibodies was mild to moderate and became stronger following chlorambucil treatment. Three patients who became resistant to VAD were found to express p-gp 170. We conclude that MDR can frequently be detected in patients with CLL. Furthermore, the expression of p-gp 170 increases with advancing stage, but not prior alkylating agent therapy. The functional expression of p-gp 170 increases with advancing stage and prior treatment and correlates well with monoclonal antibody detection (especially MRK-16). Patients who become resistant to VAD more frequently express p-gp 170 by a variety of techniques. PSC-833 is a more potent modulator of MDR than cyclosporin-A (CsA) ex vivo, and correlates better with stage of disease.

Effects of Climate and Sewer Condition on Virus Transport to Groundwater.

Pathogen contamination from leaky sanitary sewers poses a threat to groundwater quality in urban areas, yet the spatial and temporal dimensions of this contamination are not well understood. In this study, 16 monitoring wells and six municipal wells were repeatedly sampled for human enteric viruses. Viruses were detected infrequently, in 17 of 455 samples, compared to previous sampling at these wells. Thirteen of the 22 wells sampled were virus-positive at least once. While the highest virus concentrations occurred in shallower wells, shallow and deep wells were virus-positive at similar rates. Virus presence in groundwater was temporally coincident, with 16 of 17 virus-positive samples collected in a six-month period. Detections were associated with precipitation and occurred infrequently during a prolonged drought. The study purposely included sites with sewers of differing age and material. The rates of virus detections in groundwater were similar at all study sites during this study. However, a relationship between sewer age and virus detections emerged when compared to data from an earlier study, conducted during high precipitation conditions. Taken together, these data indicate that sewer condition and climate affect urban groundwater contamination by human enteric viruses.

Human and Bovine Viruses and Bacteria at Three Great Lakes Beaches: Environmental Variable Associations and Health Risk

Waterborne pathogens were measured at three beaches in Lake Michigan, environmental factors for predicting pathogen concentrations were identified, and the risk of swimmer infection and illness was estimated. Waterborne pathogens were detected in 96% of samples collected at three Lake Michigan beaches in summer, 2010. Samples were quantified for 22 pathogens in four microbial categories (human viruses, bovine viruses, protozoa, and pathogenic bacteria). All beaches had detections of human and bovine viruses and pathogenic bacteria indicating influence of multiple contamination sources at these beaches. Occurrence ranged from 40 to 87% for human viruses, 65-87% for pathogenic bacteria, and 13-35% for bovine viruses. Enterovirus, adenovirus A, Salmonella spp., Campylobacter jejuni, bovine polyomavirus, and bovine rotavirus A were present most frequently. Variables selected in multiple regression models used to explore environmental factors that influence pathogens included wave direction, cloud cover, currents, and water temperature. Quantitative Microbial Risk Assessment was done for C. jejuni, Salmonella spp., and enteroviruses to estimate risk of infection and illness. Median infection risks for one-time swimming events were approximately 2 × 10(-5), 8 × 10(-6), and 3 × 10(-7) [corrected] for C. jejuni, Salmonella spp., and enteroviruses, respectively. Results highlight the importance of investigating multiple pathogens within multiple categories to avoid underestimating the prevalence and risk of waterborne pathogens.