Direct detection of Helicobacter pylori resistance to macrolides by a polymerase chain reaction DNA enzyme immunoassay in gastric biopsy specimens

Background-The increasing use of macrolides especially in the treatment of Helicobacter pylori infection has led to an increase in resistant strains. The resistance of H pylori to macrolides, especially clarithromycin, is one of the major causes of eradication failure. In H pylori, clarithromycin re sistance is due to point mutations localised in domain V of 23S rRNA. Aim-To develop a molecular technique based on amplification of a relevant f ragment of the 23S rRNA and colorimetric hybridisation in liquid phase to d etect directly in biopsy specimens the type of mutation associated with res istance of H pylori to clarithromycin. Methods-Gastric biopsy samples from 61 patients were submitted to this test . The results were compared with standard methods (determination of minimal inhibition concentration, polymerase chain reaction/restriction fragment l ength polymorphism, and/or DNA sequencing) in order to evaluate the test an d to define the cut off values, specificity, and sensitivity. Results-The 14 biopsy samples in which H pylori was not detected did not gi ve a positive result in any assay, and the 14 samples harbouring strains su sceptible to clarithromycin gave a positive result with the wild type probe as expected. The 33 biopsy specimens containing resistant strains always g ave a positive signal with one of the probes detecting resistant organisms, but in eight cases they also reacted with the wild type probe, indicating that a mixture of resistant and susceptible organisms was present. Conclusion-The importance of this new assay is that it allows the detection of multiple genotypes corresponding to either heterogeneous genotypes or m ixed infections. Moreover, it allows in a single step not only the detectio n of H pylori but also the determination of its susceptibility to clarithro mycin directly in biopsy specimens without the need for culture.