Rapid detection of clarithromycin resistance in Helicobacter pylori using a PCR-based denaturing HPLC assay

Objectives: We evaluated a new approach for the rapid detection of clarithromycin resistance in Helicobacter pylori, based on PCR and denaturing HPLC (DHPLC). Methods: A 180 bp fragment of the 23S rRNA gene was amplified using DNA from 81 clinical H. pylori isolates (51 isolates were shown to be resistant to clarithromycin by Etest), and, directly, from 101 gastric biopsies from patients with digestive diseases, who were infected with H. pylori as assessed by a C-13-urea breath test, histology and/or culture. DHPLC was used to detect mutations in all the PCR products. Results: DHPLC profiles for the 30 susceptible isolates all showed homoduplex peaks; the resistant isolates consistently generated heteroduplex peaks that were easily distinguishable from the wild-type H. pylori reference strain. Sequencing revealed point mutations in all the resistant isolates. Overall, five different mutations were detected. Four of these mutations (A2142G, A2142C, A2143G and T2182C) are known to be associated with clarithromycin resistance; the remaining mutation (C2195T) has not been previously described. This novel single-base substitution was found in combination with the common mutation A2143G. Of the biopsies tested, 25 specimens generated heteroduplexes due to sequence alterations (mutation A2142G, A2142C or A2143G). In one of these specimens, A2143G was found together with the novel mutation T2221C; in another, a mixture of wild-type and mutant (A2143G) sequences was detected. For 20 culture-positive out of the 25 biopsies DHPLC results confirmed the presence of clarithromycin resistance. Conclusions: Our results suggest that the PCR-DHPLC assay is a valid tool for rapid assessment of clarithromycin resistance in H. pylori and that in the future it could be used directly on biopsy specimens, avoiding the need for culture-based methods.