United States Environmental Protection Agency Office of Research and Development
National Exposure Research Laboratory
Research Abstract
Government Performance Results Act (GPRA) Goal #2.3.2
Annual Performance Measure # 206
Significant Research Findings:
Improved method(s) for CCL-related microbes for use in
Unregulated Contaminant Monitoring Rule (UCMR)
Scientific Many human pathogenic viruses can cause disease in individuals who are exposed
Problem and to inadequately treated drinking water or to recreational waters contaminated with
Policy Issues fecal waste. For example, enteroviruses are commonly found in U.S. surface
waters and can cause a range of diseases including diabetes, encephalitis, eye
infections, gastroenteritis, hepatitis, meningitis, myocarditis, and paralysis.
Similarly, caliciviruses and rotaviruses cause gastroenteritis and can be transmitted
by water. The Unregulated Contaminant Monitoring Rule (UCMR) was
established to obtain occurrence data so the Office of Water can assess risk and
make regulatory determinations for microbes and chemicals on the Contaminant
Candidate List (CCL). To date, however, waterborne viruses have not been
included in the UCMR due to the lack of validated methods to detect a broad range
of these pathogens.
Research This research abstract describes three studies designed to test virus method
Approach improvements and performance over a range of water types. Specifically, these
studies describe (1) the use of EPA methods to detect waterborne virus during an
outbreak, (2) the development and testing of an assay that is able to detect a wide
variety of viruses, and (3) the design of internal controls that are able to more
easily detect when measurements of viruses by polymerase chain reaction (PCR)
are likely to be inhibited and, therefore, be artificially low.
The first study field tested EPA's method to detect caliciviruses in outbreak
settings (Anderson el al., 2003). Scientists from the National Exposure Research
Laboratory worked with those from EPA Region VIII, the Centers for Disease
Control and the Wyoming Department of Health to investigate a drinking water
outbreak that occurred in central Wyoming. Viruses were collected from the
outbreak-associated drinking water source by concentrating a sample of 2,000
liters on a positively charged cartridge filter. Eluted virus was measured using a
polymerase chain reaction (PCR) technique designed to detect the majority of
noroviruses, which are the group of caliciviruses that are known to cause
waterborne disease outbreaks.
The second study tested both the behavior of a molecular method for
enteroviruses, hepatitis A virus, noroviruses, reoviruses, and rotaviruses in a
variety of surface water types and the benefit of a control for the measurement of
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virus recovery. The study evaluated whether or not a method that had been
designed for detecting viruses in groundwaters would also be useful for detecting
viruses in more turbid surface waters. This was done by testing surface water sites
that are part of the U.S. Geological Survey's National Water Quality Assessment
(NAWQA) Program. Each of five sites was tested three times with one site being
sampled after a rain event when the streams would contain higher levels of
sewage, organic material and minerals.
The study also evaluated the efficiency with which the sample collection methods
are able to collect viruses from real world water samples. When viruses are
collected from ground and surface waters by filtration through positively charged
filters, the recovery rates may be severely impacted by water quality factors, such
as mineral content (e.g., negatively charged ions of salts), organic compounds
(e.g., humic acids) and pH. However, since measurement of recovery in the field
is rarely reported, interpreting virus occurrence data is difficult. Recovery studies
are not done routinely because of the difficulty in both transporting live virus into
the field without loss of infectivity and in preventing the seed virus from
contaminating the environment. In this study, virus recovery was determined by
collecting unseeded and virus-seeded samples at each sampling event using a
procedure that was simple and that did not contaminate the environment. This was
done by injecting a vial of Sabin poliovirus vaccine stock into the water before the
filter and by collecting the water that passed through the filter into a metal drum
containing disinfectant.
The third study developed internal control standards that can be used to test for
PCR inhibition. Environmental waters often contain chemicals that can inhibit
PCR and produce false-negative PCR results. Methods to detect viruses in water
normally contain procedures to reduce the inhibition, but these are not equally
effective in all water types. Due to this variability, all samples tested by PCR must
include a control to demonstrate whether or not inhibitors are removed. This
control is typically a virus-seeded sample; however, this requires that each sample
be analyzed twice. Internal control standards reduce labor and reagent costs by
allowing the control to be analyzed along with viruses of interest in the same
reaction tube. The internal standards designed in this study are RNA molecules
that are identical to the viruses being detected, except for a small modification in
the region used to detect the virus through PCR amplification. The modification is
designed to produce a PCR product that is smaller than that from the parent virus
genome. This allows the amplified internal control to be distinguished from the
amplified virus product by electrophoresis. The internal standards are made by
genetically modifying PCR fragments and cloning them into DNA expression
vectors. The expression vectors have sequences that can be used to produce large
quantities of RNA molecules, which are then purified and used as the internal
control standards.
Results and
Impact
The first study confirmed the utility of EPA s method for detecting noroviruses in
drinking water during an outbreak investigation. An outbreak of gastroenteritis
affecting at least 84 patrons had occurred in a saloon in central Wyoming during
September and October, 2001. The investigation demonstrated that the ground
water well being used by the saloon was impacted by septic system sewage and
that a water chlorinator had failed during the time of the outbreak. Water collected
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from the well was positive for fecal coliforms. It also contained the same strain of
norovirus that was found in patients affected from the outbreak.
The second study showed that an EPA molecular method designed for virus
detection in ground water could be used with surface water. NAQWA sites in
Washington State, Iowa, Ohio, West Virginia and South Carolina were selected
based upon geographic location, population density and land usage (agricultural,
mining, urban, etc.). Population densities of the chosen sites ranged from 16 to
15,540 individuals per square kilometer. Water quality parameters were typical
for streams around the US. Water temperatures ranged from 0 to 12.6°C,
reflecting the winter and spring sampling conditions, and pH values ranged from
5.4 to 8.1. Turbidity ranged from 0.7 to 344 nephelometric turbidity units and E.
coli concentrations ranged from 59 to 23,000 colony-forming units per 100 mL.
Each site was tested three times for viruses using cultural and molecular methods.
All sites were positive for viruses by both methods. A total of 87% of the samples
were positive for enteroviruses, 30% for reoviruses, 20% for rotaviruses and 17%
for hepatitis A virus. Quality control data were critical in the interpretation of the
molecular results. Based in part on the results from this study, a number of quality
controls that are not normally considered were recommended for future virus
occurrence studies, and these recommendations have been incorporated into a joint
Office of Water/Office of Research and Development guidance document entitled,
"Quality Assurance/Quality Control Guidance for Laboratories Performing PCR
Analyses on Environmental Samples." Infectious enteroviruses were found in four
samples collected from streams which would have met EPA's guidelines for safe
recreational water usage. This suggests that indicator levels that are at or below
the guidelines do not always predict the viral quality of waters.
This study also demonstrated that the procedure to measure virus recoveries could
be performed safely in the field. Poliovirus recoveries from stream water averaged
45% and ranged from 16 to 65%. Large recovery differences were observed
between individual samples at two of the sites and recovery rates did not appear to
be inversely correlated to turbidity as generally assumed. Unfortunately, with the
limited number of samples in this study, determining specific correlations between
water quality and virus recovery was not possible. However, the importance of
virus recovery controls was suggested by the detection of a greater number of
virus types in all samples with higher virus recoveries, with the possible exception
of samples from the West Virginia site.
The third study describes the development and testing of internal controls for
enteroviruses, hepatitis A virus (HAV), Norwalk virus and rotaviruses. The
controls were shown by gene sequencing to have only small differences between
the size and the sequence of the control and that of the respective virus. It was
shown that each control could be distinguished from the virus for which it was
designed by electrophoresis and by hybridization with specific probes. Procedures
to produce internal standards comprised of RNA (free of DNA from the expression
vector) were developed and were successfully used to demonstrate the absence of
inhibitors from environmental water samples. These controls not only reduce
labor and reagent costs, but they also are thought to decrease the number of false
positive reactions by eliminating the need to run virus-seeded samples.
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The results of this research support the Government Performance and Results Act
Goal 2 ("Clean and Safe Water"), Sub-Objective 2.3.2 ("Conduct Leading-Edge
Research"), and Long Term Goal DW-3 ("Unregulated Contaminants and
Innovative Approaches, By FY 2010, develop new data, innovative tools and
improved technologies to support decision making by the Office of Water on the
Contaminant Candidate List and other regulatory issues, and implementation of
rules by states, local authorities and water utilities"). The research was done in
support of an FY05 annual performance goal #131 ("Provide the Office of Water
with the results of health effects, exposure / methods and treatment studies, in
support of decisions to regulate or not regulate at least five pathogens and toxins
on the Contaminant Candidate List.") and annual performance measure #206
("Improved method(s) for CCL-related microbes for use in Unregulated
Contaminate Monitoring Rule (UCMR), e.g. enteroviruses, caliciviruses,
rotaviruses").
A portion of this research was performed under an Interagency Agreement with
the U.S. Geological Survey.
The findings of this research have been published (Publication No. NERL-CI-
MCEARD-03-003, NERL-CI-MCEARD-03-032, NERL-CI-MCEARD-03-092,
respectively):
Parshionikar, S.U., Willian-True, S., Fout, G.S., Robbins, D.E., Seys, S.A.,
Cassady, J.D., and Harris, R. "Waterborne outbreak of gastroenteritis associated
with a norovirus." AppL Environ. Microbiol. 69:5263-8, 2003.
Denis-Mize, K., Fout, G.S., Dahling, D.R., and Francy, D.S. "Detection of
Human Enteric Viruses in Stream Water with PCR and Cell Culture." J. Water
Health. 2:37-47, 2004.
Parshionikar, S.U. Cashdollar, J., and Fout, G.S. "Development of homologous
viral internal controls for use in RT-PCR assays of waterborne enteric viruses." J.
Virol. Methods, 121: 39-48, 2004.
Future Research Additional improvements are still needed before the methods can be used for
routine water monitoring for viruses. The highest priority areas for needed
improvements are the development of an alternative to the expensive positively
charged 1MDS filter and the development of techniques to define human health
risks based upon molecular assays.
Questions and inquiries can be directed to:
G. Shay Fout, Ph.D.
US EPA, Office of Research and Development
National Exposure Research Laboratory
Cincinnati, OH 45268
Phone: 513/569-7387
E-mail: fout.shav@epa.gov
Research
Collaboration and
Research
Products
Contacts for
Additional
Information
Donna S. Francy
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U.S. Geological Survey
6480 Doubletree Ave.
Columbus, OH 43229
Phone: 614/430-7769
E-mail: dsfrancv@usgs.gov
Sandhya Parshionikar, Ph.D.
US EPA, Office of Water
Technical Services Center
Cincinnati, OH 45268
Phone: 513/569-7123
E-mail: parshionikar.sandhya@epa.gov
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