Detection of Helicobacter pylori and clarithromycin resistance in gastric biopsies of pediatric patients by using a commercially available real-time polymerase chain reaction after NucliSens semiautomated DNA extraction

Abstract

The aim of this study was to evaluate a commercially available kit, MutaREAL Helicobacter pylori (Inmundiagnostik, Bensheim, Germany) real-time polymerase chain reaction (PCR), for detection of H. pylori infection and point mutations in the 23S rRNA genes responsible for clarithromycin resistance in gastric biopsies.

Methods: Gastric biopsies were obtained by endoscopy from pediatric patients with gastric symptoms, cultured according to standard microbiologic procedures, and clarithromycin resistance was determined by E-test. DNA extraction was performed by NucliSens platform with the NucliSens magnetic extraction reagents (bioMérieux, Marcy-l'Etoile, France) according to the manufacturer's instructions. MutaREAL kit was used according to manufacturer recommendations in a LightCycler (Roche Diagnostics Gmbh, Mannheim, Germany) for the detection of H. pylori infection and clarithromycin susceptibility.

Results: Amplification was positive for H. pylori in 62 and negative in 44 biopsies out of 106 biopsies. All negative biopsies were positive for human beta-globin gene. This real-time PCR assay showed sensitivity of 93.33% (negative predictive value, 90.90%) and specificity of 86.95% (positive predictive value, 90.32%) for H. pylori detection. Clarithromycin resistance was detected in 26 cases by PCR with a sensitivity and specificity of 90.62 and 95.83, respectively.

Conclusions: MutaREAL kit was able to detect H. pylori and its clarithromycin susceptibility with high efficacy. This method is quicker than culture and is suitable to be done in 1 h after DNA extraction. The new system of automatic extraction will lead to reduction in the total time.