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 β-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.

Keywords: Clarithromycin, Biopsies, Real-time PCR, Resistance and easyMAG