G. Shay Fout

Waterborne Outbreak of Gastroenteritis Associated with a Norovirus

ABSTRACT The Wyoming Department of Health investigated an outbreak of acute gastroenteritis among persons who dined at a tourist saloon in central Wyoming during October 2001. Human caliciviruses (HuCVs) were suspected as the etiological agent of the outbreak based on the incubation period, duration of illness, and symptoms observed in ill patrons. A retrospective cohort study demonstrated that ill patrons were 4.5 times more likely to have exposure to drinking water and/or ice than nonill patrons. No food items were associated with illness. An environmental investigation gave evidence that the saloons groundwater was contaminated with sewage. Water from the saloons only well was processed for viruses. The processed water sample and stool samples collected from three ill patrons were analyzed by reverse transcription-PCR (RT-PCR) for the presence of HuCV. All positive RT-PCR results were confirmed by sequence and phylogenetic analyses of cloned RT-PCR products. A genogroup I, subtype 3, HuCV stain was found to be present in the well water sample and two stool samples. In addition, a genogroup II, subtype 6, strain was detected in one stool sample. The identification of the same HuCV strain in both the well water and stool samples strongly suggests a link between exposure to well water and the outbreak of gastroenteritis. The presence of a genogroup II, subtype 6, strain in one of the stool samples suggests that multiple HuCV strains may have been involved in this outbreak. The laboratory isolation of HuCV strains from outbreak-associated drinking water is relatively novel in the United States. This investigation outlines the procedure for virus isolation and illustrates the utility of RT-PCR for the identification of HuCV in large volumes of water and stool samples obtained during outbreaks of acute nonbacterial gastroenteritis.

A Multiplex Reverse Transcription-PCR Method for Detection of Human Enteric Viruses in Groundwater

ABSTRACT Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States. Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viruses found in groundwater. To address this problem, monthly samples from 29 groundwater sites were analyzed for 1 year for enteroviruses, hepatitis A virus, Norwalk virus, reoviruses, and rotaviruses by multiplex reverse transcription-PCR (RT-PCR). A procedure with which to remove environmental RT-PCR inhibitors from groundwater samples was developed. The procedure allowed an average of 71 liters of the original groundwater to be assayed per RT-PCR, with an average virus recovery rate of 74%, based on seeded samples. Human enteric viruses were detected in 16% of the groundwater samples analyzed, with reoviruses being the most frequently detected virus group.

Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples

ABSTRACT Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.

New Electropositive Filter for Concentrating Enteroviruses and Noroviruses from Large Volumes of Water

ABSTRACT The U.S. Environmental Protection Agencys information collection rule requires the use of 1MDS electropositive filters for concentrating enteric viruses from water, but unfortunately, these filters are not cost-effective for routine viral monitoring. In this study, an inexpensive electropositive cartridge filter, the NanoCeram filter, was evaluated for its ability to concentrate enteroviruses and noroviruses from large volumes of water. Seeded viruses were concentrated using the adsorption-elution procedure. The mean percent retention of seeded polioviruses by NanoCeram filters was 84%. To optimize the elution procedure, six protocols, each comprising two successive elutions with various lengths of filter immersion, were evaluated. The highest virus recovery (77%) was obtained by immersing the filters in beef extract for 1 minute during the first elution and for 15 min during the second elution. The recovery efficiencies of poliovirus, coxsackievirus B5, and echovirus 7 from 100-liter samples of seeded tap water were 54%, 27%, and 32%, respectively. There was no significant difference in virus recovery from tap water with a pH range of 6 to 9.5 and a water flow rate range of 5.5 liters/min to 20 liters/min. Finally, poliovirus and Norwalk virus recoveries by NanoCeram filters were compared to those by 1MDS filters, using tap water and Ohio River water. Poliovirus and Norwalk virus recoveries by NanoCeram filters from tap and river water were similar to or higher than those by the 1MDS filters. These data suggest that NanoCeram filters can be used as an inexpensive alternative to 1MDS filters for routine viral monitoring of water.

A Waterborne Outbreak of Norwalk-Like Virus among Snowmobilers—Wyoming, 2001

In February 2001, episodes of acute gastroenteritis were reported to the Wyoming Department of Health from persons who had recently vacationed at a snowmobile lodge in Wyoming. A retrospective cohort study found a significant association between water consumption and illness, and testing identified Norwalk-like virus (NLV) in 8 of 13 stool samples and 1 well. Nucleotide sequences from the positive well-water specimen and 6 of the positive stool samples were identical. This multistrain NLV outbreak investigation illustrates the importance of NLV as a cause of waterborne illness and should encourage monitoring for NLVs in drinking water.

A method to remove environmental inhibitors prior to the detection of waterborne enteric viruses by reverse transcription-polymerase chain reaction

A method was developed to remove environmental inhibitors from sample concentrates prior to detection of human enteric viruses using the reverse transcription-polymerase chain reaction (RT-PCR). Environmental inhibitors, concentrated along with viruses during water sample processing, are removed by the method through a series of steps that includes dialysis, solvent extraction, ultrafiltration and glass purification. The method was tested by spiking sodium phosphate with poliovirus type 1 with or without humic or fulvic acids and then measuring virus recovery by plaque assay and RT-PCR. Results of the study indicated that (i) 90% of the spiked virus could be recovered from samples at the end of the ultrafiltration step, (ii) virus was detected in the final eluate of samples containing as much as 0.5 mg of humic acid or 5.0 mg of fulvic acid, and (iii) as little as 0.06 plaque forming units (PFU) was detectable per RT-PCR reaction. These results indicate that the described purification method along with RT-PCR is a feasible approach for detecting waterborne human enteric viruses in the presence of interfering substances.

Survey of ground, surface, and potable waters for the presence of Legionella species by EnviroAmpR PCR Legionella Kit, culture, and immunofluorescent staining

Abstract A total of 116 samples from numerous aquatic sources including water from faucets, showerheads, dental units, fire sprinklers, and surface waters were examined for the presence of Legionella by the EnviroAmpR Legionella PCR Kit, culture on BCYEα, or direct fluorescent antibody (DFA) staining. In addition, 58 ground water samples collected across the U.S. were also examined for the presence of Legionella species by PCR and by culture on BCYEα and BCYEα-GVPC. Of the 58 ground water samples tested by polymerase chain reaction (PCR) assay, 93% were positive by PCR, while only 7% were culture positive. Analysis of the remaining samples indicated that 52.7% of the samples were positive by PCR, 50.0% were positive by DFA, while 8.3% were culture positive. Although Legionella species were frequently detected by PCR in all water types examined, concentrations were typically less than 103 cells per ml. Legionalla species were detected much more frequently than was Legionella pneumophila in all aquatic samples. DFA results correlated well to PCR results but culture results were often negative for Legionella due to overgrowth, competition, or inhibition by background organisms. Results of this type of survey confirm the ubiquity of Legionella in aquatic environments, even ground waters. This study represents the first in depth survey of ground water across the United States.

Development and Evaluation of EPA Method 1615 for Detection of Enterovirus and Norovirus in Water

ABSTRACT The U.S. EPA developed a sample concentration and preparation assay in conjunction with the total culturable virus assay for concentrating and measuring culturable viruses in source and drinking waters as part of the Information Collection Rule (ICR) promulgated in 1996. In an effort to improve upon this method, the U.S. EPA recently developed Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Method 1615 uses a culturable virus assay with reduced equipment and labor costs compared to the costs associated with the ICR virus method and introduces a new molecular assay for the detection of enteroviruses and noroviruses by reverse transcription-quantitative PCR. In this study, we describe the optimization of several new components of the molecular assay and examine virus recovery from ground, reagent-grade, and surface water samples seeded with poliovirus type 3 and murine norovirus. For the culturable virus and molecular assays, mean poliovirus recovery using the complete method was 58% and 20% in groundwater samples, 122% and 39% using low-titer spikes in reagent-grade water, 42% and 48% using high-titer spikes in reagent-grade water, and 11% and 10% in surface water with high turbidity, respectively. Murine norovirus recovery by the molecular assay was 30% in groundwater samples, less than 8% in both low- and high-titer spikes in reagent-grade water, and 6% in surface water with high turbidity. This study demonstrates the effectiveness of Method 1615 for use with groundwater samples and highlights the need for further research into its effectiveness with surface water.

Development of a multiplex microsphere immunoassay for the quantitation of salivary antibody responses to selected waterborne pathogens.

Saliva has an important advantage over serum as a medium for antibody detection due to non-invasive sampling, which is critical for community-based epidemiological surveys. The development of a Luminex multiplex immunoassay for measurement of salivary IgG and IgA responses to potentially waterborne pathogens, Helicobacter pylori, Toxoplasma gondii, Cryptosporidium, and four noroviruses, involved selection of antigens and optimization of antigen coupling to Luminex microspheres. Coupling confirmation was conducted using antigen specific antibody or control sera at serial dilutions. Dose-response curves corresponding to different coupling conditions were compared using statistical tests. Control proteins in the specific antibody assay and a separate duplex assay for total immunoglobulins G and A were employed to assess antibody cross-reactivity and variability in saliva composition. 200 saliva samples prospectively collected from 20 adult volunteers and 10 paired sera from a subset of these volunteers were used to test this method. For chronic infections, H. pylori and T. gondii, individuals who tested IgG seropositive using commercial diagnostic ELISA also had the strongest salivary antibody responses in salivary antibody tests. A steep increase in anti-norovirus salivary antibody response (immunoconversion) was observed after an episode of acute diarrhea and vomiting in a volunteer. The Luminex assay also detected seroconversions to Cryptosporidium using control sera from infected children. Ongoing efforts involve further verification of salivary antibody tests and their application in larger pilot community studies.

Differential Decay of Enterococci and Escherichia coli Originating from Two Fecal Pollution Sources

ABSTRACT Using in situ subtropical aquatic mesocosms, fecal source (cattle manure versus sewage) was shown to be the most important contributor to differential loss in viability of fecal indicator bacteria (FIB), specifically enterococci in freshwater and Escherichia coli in marine habitats. In this study, sunlight exposure and indigenous aquatic microbiota were also important contributors, whose effects on FIB also differed between water types.