Pyrosequencing analysis for rapid and accurate detection of clarithromycin resistance-associated mutations in Iranian Helicobacter pylori isolates

Abstract

Background: Treatment of Helicobacter pylori (H. pylori) infection has become challenging following the development of primary antibiotic resistance. A primary therapeutic regimen for H. pylori eradication includes clarithromycin; however, the presence of point mutations within the 23S rRNA sequence of H. pylori contributes to clarithromycin resistance and eradication failure. Thus, we aimed to develop a rapid and precise method to determine clarithromycin resistance-related point mutations using the pyrosequencing method. Methods and results: H. pylori was isolated from 82 gastric biopsy samples and minimal inhibitory concentration (MIC) was evaluated using the agar dilution method. Clarithromycin resistance-associated point mutations were detected by Sanger sequencing, from which 11 isolates were chosen for pyrosequencing. Our results demonstrated a 43.9% (36/82) prevalence in resistance to clarithromycin. The A2143G mutation was detected in 8.3% (4/48) of H. pylori isolates followed by A2142G (6.2%), C2195T (4.1%), T2182C (4.1%), and C2288T (2%). Although the C2195T mutation was only detected by Sanger sequencing, the overall results from pyrosequencing and Sanger sequencing platforms were comparable. Conclusions: Pyrosequencing could be used as a rapid and practical platform in clinical laboratories to determine the susceptibility profile of H. pylori isolates. This might pave the way for efficient H. pylori eradication upon detection.