Genotyping Helicobacter pylori from faecal DNA
Abstract number: P1695
McMillan M., MacKay W.G., Williams C.L., Weaver L.T.
Objectives:Helicobacter pylori is the commonest chronic bacterial infection of mankind. It is usually acquired during childhood and in the developed world it is the principal cause of duodenal ulcer disease and a risk factor for gastric cancer. In the developing world it is associated with undernutrition and short stature. Non-invasive methods of diagnosis and detection involve the urea breath test, serology and faecal antigen analysis. However genotyping bacterial isolates from children could enhance epidemiological investigations into routes of transmission of this microorganism. This study exploits the excretion of H. pylori in the faeces to obtain H. pylori DNA for genotyping and thereby to generate dendograms to explore patterns of intrafamilial transmission.
Methods: Subjects were children (<16y) attending the Royal Hospital for Sick Children in Glasgow for urea breath test for suspected H. pylori infection. Each subject, and close family members (parents and sibs), was asked to provide a stool sample, which was stored at -80°C until analysis. Twenty-eight stool samples that were positive by HpSA faecal antigen test (Meridian Diagnostics Inc) were analysed. H. pylori DNA was recovered using phenol: chloroform: isoamylalcohol and ethanol precipitation followed by isolation using a specific biotinylated oligonucleotide probe with magnetic capture. RAPD PCR  was used as a means of genotyping H. pylori DNA isolated from faeces.
Results: Data from sixteen families were analysed. One family had four members, one had three members, seven had two members and seven had one member. RAPD analysis revealed a typeability of ninety-three percent. Loose similarities were observed between family one involving mother and children but not between father and children. Similarly in two other families samples from mother and child bore similarities. All parent samples and those from other families bore little correlation.
Conclusion: Isolation of purified H. pylori by gene capture offers a means of extracting DNA from faecal samples which can be use to genotype the microorganism. The majority of faecal H. pylori isolates were successfully genotyped using RAPD which proved to be highly discriminating.
1. Akopyanz, N., N.O. Bykanov, T.U. Westblom, S. Kresovich and D.E. Berg. 1992. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res. 20. 51375142.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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