Sequencing-based detection of 23S rRNA domain V mutations in treatment-naïve Helicobacter pylori patients from Malaysia

OBJECTIVES: In Malaysia, the prevalence of Helicobacter pylori resistance to clarithromycin is increasing. This study aimed to determine mutations in the 23S rRNA domain V directly using bacterial DNA extracted from gastric biopsy specimens with a urease-positive result.

METHODS: A 1085-bp fragment of 23S rRNA domain V from samples of 62 treatment-naïve patients with H. pylori infection was amplified by PCR with newly designed primers, followed by sequencing.

RESULTS: Of the 62 cases, 42 patients were treated with clarithromycin-based triple therapy and 20 patients were treated with amoxicillin and proton pump inhibitor only; both therapies showed successful eradication rates of 70-73.8%. Sequencing analysis detected 37 point mutations (6 known and 31 novel) with prevalences ranging from 1.6% (1/62) to 72.6% (45/62). A2147G (aka A2143G) appears to be associated with a low eradication rate [40% (2/5) failure rate and 13.3% (6/45) treatment success rate], supporting its role as a clinically significant point mutation. T2186C (aka T2182C) was found in 71.1% (32/45) and 80% (4/5) of treatment success and failure cases, respectively, suggesting that the mutation is clinically insignificant. The eradication success rate in patients with the novel T2929C mutation was decreased three-fold (6.7%; 3/45) compared with the failure rate (20%; 1/5), suggesting that it may play an important role in clarithromycin resistance, thus warranting further study.

CONCLUSION: This study identified multiple known and novel mutations in 23S rRNA domain V through direct sequencing. Molecular detection of clarithromycin resistance directly on biopsies offers an alternative to conventional susceptibility testing.