Development of polymerase chain reaction coupled with a high-resolution melting technique for detection of Helicobacter pylori clarithromycin resistance

Abstract

Helicobacter pylori, a bacterial pathogen that causes severe gastric diseases, has developed antibiotics resistance; mainly against clarithromycin. Consequently, the latest international recommendations have advised clarithromycin susceptibility testing before prescribing the first line of therapy. This resistance is predominantly related to the A2143G point mutation of 23S rRNA. The aim of this study was to develop a highly sensitive and cheap molecular technique to detect H. pylori clarithromycin resistant strains. For that, plasmids of H. pylori 23S rRNA region harboring A2143G point mutation were constructed and used to develop polymerase chain reaction coupled with a high-resolution melting technique (PCR-HRM). Then, this method was applied on 233 gastric H. pylori positive samples. The obtained results showed that the developed PCR-HRM technique allowed specific identification of clarithromycin resistant and heteroresistant H. pylori samples, even if the ratio of resistant/ sensitive samples was low. In this series, the detected rate of H. pylori clarithromycin resistance was 7.6 %, which concord with the resistance rate determined using molecular sequencing. So, the developed PCR-HRM represents a good tool for detecting resistant and sensitive H. pylori samples and can be used in routine clinical practices. This will help in managing H. pylori infection, increase its eradication rate, and avoid the use of therapeutic protocols that have serious side effects.

Keywords

• h. pylori
• pcr-hrm
• 23s rrna
• clarithromycin resistance
• hetero-resistance