Clarithromycin resistance assessment in Helicobacter pylori isolates by using 23S rRNA gene molecular markers

Background: H. pylori is a relatively fastidious and microaerophilic microorganism and therefore standard phenotypic susceptibility tests, even in the hands of experts, are slow and can take at least 10 14 days. Molecular based diagnostic assays by using molecular markers for resistance detection offer an attractive alternative approach to obtain susceptibilities to antibiotics with greater accuracy and speed, and the possibility of a same day result. The aim of this study is the assessment of clarithromycin resistance by using molecular markers. Methods: This cross-sectional descriptive study was performed on 200 gastric biopsy specimens which were obtained from patients undergoing upper gastrointestinal tract endoscopy in Hajar hospital of Shahrekord, by using TaqMan real-time PCR. Initially, H. pylori strains were identified by RUT and PCR. Then, by this regard that accumulation of mutations associated with resistance to clarithromycin were in the region between nucleotides 2142 2144 of 23S rRNA gene, the first probe was designed to be able the distinguish between sensitive and resistant strains. Finally four probes were designed that each be able to identify only one mutation associated with a particular level of clarithromycin resistance. Results: Out of 200 samples, 164 (82%) were H. pylori positive. Overall, clarithromycin susceptible strains were detected in 105 (64.02%) patients and clarithromycin resistance were detected in 59 (35.98%) which were identified as 4 (2.44%) A2144G, 26 (15.85%) A2143G, 15 (9.15%) A2143C and 20 (12.19%) A2142G point mutations. Purely resistant strains were detected in 38 (23.17%), while heteroresistant were found in the remaining 16 (9.76%) cases. Genotype of 5 (8.47%) strains was not detected. This data was confirmed by PCR-RFLP technique. Conclusion: Results showed that Real-time PCR assay in combination with molecular markers has high accuracy to simultaneously identify H. pylori and clarithromycin resistance types directly in gastric biopsy specimens in short time.