Rapid and accurate detection of point mutations conferring resistance to clarithromycin in Helicobacter pylori by real-time PCR

Abstract

The main cause of failure of Hclicobacter pylori eradication therapy is resistance to clarithromycin. The resistance is due to point mutations in 2 positions on the 23S rRNA. Our aim was to develop a rapid and accurate method to detect these mutations directly on biopsy specimens. We developed a real-time PCR assay including a simultaneous detection of the amplicons by hybridization of two probes labelled with LC-Red and flurorescein using the Fluorescence Resonance Energy Transfer technology. The assay was first apphed successfully on reference strains, reference plasmids, and H. pylori negative biopsies. Then biopsies from 200 patients having failed a first eradication attempt and for whom the Helicobacter pylori strain was available were tested with the new assay. A result was obtained in 199 cases, a single genotype was detected in 157 cases, two genotypes in 41 cases, and three genotypes in one case. There were in total 7 discrepancies between the real-time PCR assay and the phenotypic methods of determination of clarithromycin susceptibility, and in an additional 4 eases the 2 phenotypie methods were in disagreement. PCR-RFLP was applied to a sampling of biopsies including all of the cases with multiple genotypes and all with discrepant results. Finally, in 4 cases with discrepant results, the real-time PCR assay detected the resistant population at a concentration so low that it could not be detected by the phenotyptc method, while in 3 cases other mutations could be involved. This assay had an accuracy at least as satisfactory as the phenotypic tests and could he performed within 2 hours, allowing it to be used before the administration of therapy in the case of a first H. pyiori eradication.