Characterization of Domain V Mutations in Clinical Isolates of Helicobacter pylori in Pakistan and Their Effect on Clarithromycin MIC

Abstract

Purpose: Clarithromycin is commonly prescribed for H. pylori infection. Domain V mutations are responsible for clarithromycin resistance. This study was aimed to characterize the clarithromycin resistance and its associated mutations in clinical isolates of H. pylori in Pakistan. Materials and Methods: Infection was diagnosed in 93 patients' biopsies using culture, rapid urease test, 16S rRNA, and vacA gene multiplex PCR. Clarithromycin resistance was assessed by the agar dilution method. Mutations were detected by PCR-RFLP using 46 (1.4 kb) domain V fragments. Sequencing was executed for 13 domain V fragments, of which 12 showed unusual amplicon size (1.2 kb) and 01 had a new MboII RFLP pattern. Results: A total of 48 (83%) strains were obtained from 58 (62.3%) PCR H. pylori-positive samples. Resistance (MIC ≥ 0.001 mg/mL) and intermediate resistance phenotype (MIC = 0.0005 mg/mL) was observed in 22 (46%), and 10 (21%) isolates, respectively. The primary resistance was found in 23 (39.6%) samples. PCR-RFLP detected A2142G, A2143G, and double mutations in 19, 04, and 01 resistant strain, respectively. Sequencing of 10 amplicons obtained from intermediated resistant strains and 03 amplicons from resistant strains showed 138 new mutations. Among them, T2182C was also seen in 04 intermediated resistant isolates, whereas A2142G, A2143G, and A2143C were observed in resistant isolates. The new MboII RFLP pattern in an intermediated resistant strains was due to A1761G mutation. Conclusion: H. pylori domain V mutations showed extensive diversity. Multiple mutations in domain V may give endurance to H. pylori against clarithromycin. Further investigations on the molecular mechanism of antibiotic resistance in H. pylori seem crucial at this stage.

Role of MIC levels and 23S rRNA mutation sites to clarithromycin in 14-day clarithromycin bismuth quadruple therapy for Helicobacter pylori eradication: A prospective trial in Beijing

“…In our study, only 2143A > G was detected, with a rate of 34.78 %. We did not detect 2142A > C or 2142A > G in any of our cases in line with the results of many other studies owing to the small sample size [ 13 , 38 , 39 ]. Notably, all patients with eradication failure had 2143A > G. Though patients with 2143A > G did not necessarily have eradication failure, 2143A > G increased the likelihood of the chances of eradication failure.…”

“…In summary, mutations of 243ins, 353ins, 381A > T, 892G > A, 1604C > T, 1704A > G, 1883C > T, 1953C > T, 2245C > T, and 2797C > T were observed in patients who experienced eradication failure, but the frequency of occurrence was infrequent, limited to only one patient. Other previously well-studied sites like 1821A > G and 2182T > C [ [12] , [13] , [14] ] were also found in our study, but their detection significance needs to be further explored. Apart from 2143A > G, no other site exhibited a significant association with eradication outcomes.…”

“…However, eradication was still successful in some patients with MIC ranging from 16 to 32 μg/mL, indicating that susceptibility testing in vitro could not fully predict susceptibility results in vivo. As many experts recommend, antibiotic susceptibility testing should be performed before treatment [ 13 ]. We believe that rather than using susceptibility results alone to guide medication regimens, confirming the MIC results of clarithromycin before treatment helps more to improve the eradication rate [ 28 ].…”

“…It was found that 10 mutation sites only occurred in eradication-failed strains, but these sites were sporadic and could not independently predict the eradication outcome. Above all, 49 sites were found to be mutated but not associated with outcome, including 1821A > G and 2182T > C [ 13 , 38 ], which has been studied many times before. Therefore, this was the first study put forward that monitoring clarithromycin-resistant mutation sites could not guide the eradication outcome of 14-day clarithromycin bismuth quadruple therapy accurately.…”

Drug Resistance Patterns of Commonly Used Antibiotics for the Treatment of Helicobacter pylori Infection among South Asian Countries: A Systematic Review and Meta-Analysis

“… Forest Plot Showing Clarithromycin Resistance. (Reference Studies: [ 15 , 20 , 21 , 22 , 23 , 24 , 25 , 27 , 28 , 29 , 30 , 31 , 32 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]). …”