Impact/Purpose:

To determine the sensitivity and precision of an inhouse real time probe and primers for Helicobacter pylori.

To determine whether drinking water from different geographical areas will contain inhibitors that will prevent or reduce the detection of Helicobaacter pylori.

Description:

Helicobacter pylori is a newly recognized human pathogen, known to cause gastric ulcers and thought to be a contributing factor in gastric cancer. Recent studies in the scientific literature, using a variety of methodologies, report the presence of H. pylori bacteria in environmental waters, including potable waters. However the lack of occurrence data generated by a standard method limits our understanding of the significance of drinking water as a transmission vehicle for this pathogen. This study proposes to evaluate, and if possible, validate two methods that have been developed by the US EPA for detecting H. pylori bacteria in environmental waters and to use these methods to explore the occurrence of these bacteria in potable waters. Both methods are based on detecting H. pylori-specific DNA sequences in a water sample. One method includes an enrichment step that allows for the determination of the presence of culturable H. pylori cells. Reliable detection methods and subsequent occurrence data will help public health officials determine the importance of potable waters in the transmission of these pathogens. Results from this study will be used by the Agency to determine whether to include H. pylori in unregulated contaminant monitoring and will be disseminated in presentations at national and regional scientific meetings and in submission of articles to peer-reviewed scientific journals.

Record Details:

Record Type:PROJECT

Start Date:10/01/2003

Projected Completion Date:09/01/2005

OMB Category:Other

Record ID: 76747

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Project Information:

Progress :The first method, referred to as real-time PCR for H. pylori, uses state-of-the-art DNA amplification technology to detect H. pylori-specific DNA targets within several hours. Results showed a linear relationship with r^2 of 0.999 for DNA from 10^ 6 to one genome equivalent and an r^2 of 0.990 for 10^2 to 10^7 cells per ml. Non-target species were not detected or were detected at approximately a 5 log lower level of sensitivity. No differences in responses were obtained from high levels of Escherichia coli added to low to high counts of H. pylori. The sensitivity for detecting 10 cells was 50% and for 40 cells was 95%. No naturally found H. pyloriwere obtained from drinking water samples obtained from 7 different geographical areas across the country. Recoveries from dosed drinking waters by white filters were equivalent to those from identical concentrations of H. pyloriby black filters where counts were obtained from fluorescent antibodies. The H. pylori probe and primers with the methods described in this study can be used to test water samples throughout the country to measure the prevalence of this bacteria which is currently on the US EPA Office of Water Candidate Contaminant List.



The second method developed for the detection of H. pylori in water utilizes an enrichment step to not only increase the number of target cells in the sample, but through this enrichment, allows a determination of the presence of culturable cells, which will be a subset of the viable cell population. The most recent results indicate that while H. pylori will grow on the membrane filters used to capture them from water, the cells appear to lose culturability after exposure to tap water. This is in contrast to exposing them to buffered water in the lab, a situation in which the cells can be readily cultured on the filters. It is unclear how effective this method will eventually be if this negative effect on the cells can not be reversed.

Relevance :Occurrence information on the bacterium Helicobacter pylori in drinking water distribution systems is not yet available. In order to produce accurate occurrence data, specific and sensitive methods must be developed and validated. The EPA Office of Water will use this occurrence data, as well as other forms of data, to construct a risk assessment analysis to determine whether Helicobacter pylori bacteria pose a significant public health risk due to exposure to drinking water and whether the levels of these organisms in drinking water need to be regulated.

Clients :Office of Water

Project IDs:

ID Code :18221

Project type :OMIS