Second-line treatment of Helicobacter pylori infection after dilution agar methods and PCR-RFLP analysis

Background: After unsuccessful first-line treatment of Helicobacter pylori infection, the percentage of clarithromycin-resistant strains has been reported as between 30% and 70% in Japan and other countries. A high prevalence of clarithromycin-resistant strains is reported to be associated with eradication failure. Aim: We examined antibiotic susceptibility testing using a combination of dilution agar methods with PCR-restriction fragment length polymorphism (RFLP) analysis. Methods: We enrolled 41 patients in whom first-line treatment with LAC (lansoprazole, amoxycillin and clarithromycin) was unsuccessful. Endoscopic biopsied specimens were used to examine antibiotic susceptibility to clarithromycin by dilution agar methods. PCR-RFLP analysis was performed to determine the presence of point mutations, which are primarily responsible for resistance to clarithromycin. Results: Clarithromycin-resistance rate after failure of the LAC regimen was 73.2%. Drug susceptibilities of three strains obtained by PCR-RFLP analysis were different from those by dilution agar methods. One strain with MIC values to clarithromycin of 0.05 μg/mL had a point mutation, A2144G. This strain was not eradicated by repeating LAC, but was eradicated by substituting metronidazole for clarithromycin. Conclusions: Dilution agar methods should be combined with PCR-RFLP analysis before second-line eradication to increase the accuracy of clarithromycin- susceptibility testing and to improve eradication efficacy.