Real-Time PCR detection and quantitation of Helicobacter pylori clarithromycin-resistant strains in archival material and correlation with Sydney classification

Sofia Gazi; Andreas Karameris; Marios Christoforou; Niki Agnantis; Theodore Rokkas; Dimitrios Stefanou

Authors Sofia Gazi, Andreas Karameris, Marios Christoforou, Niki Agnantis, Theodore Rokkas, Dimitrios Stefanou.

Abstract

Background Helicobacter pylori (H. pylori), infects gastric mucosa causing gastritis. Treatment failure is mainly due to certain genetic changes in the peptidyltransferase loop of 23S rRNA of the microorganism. The aim of the study was to evaluate genetic changes in gastric biopsies of H. pylori (+) patients that lead to clarithromycin resistance and to correlate them with histology data.

Methods A total of 150 H. pylori (+) gastric biopsies were studied, taken before and after eradication therapy from 75 dyspeptic patients divided in 2 groups: group A consisted of 25 H. pylori (+) triple-therapy resistant patients and group B consisted of 50 H. pylori (+) successfully treated patients. Histological classification of the H. pylori (+) gastritis was done according to the Sydney criteria. Genetic material was analyzed with the ClariResTM RT-PCR bi-probe based assay for the determination of point mutations in the 23S rRNA gene and with a Quantitative-RT-PCR (Q-RT-PCR) method for the quantitation of H. pylori.

Results We showed that in 18/ 25 group A patients certain point mutations of 23S rRNA at sites A2142C, A2142G and A2143G had occurred. Nine of these 18 mutated cases (50%) were characterized as mixed infections. Mixed infections in 2/50 patients of group B were also observed. Using Q-RT-PCR, we found that gastric mucosal density of H. pylori correlates well with bacterial colonization. There was a statistically significant association (P<0.005) between the presence of the detected H. pylori genetic alterations and inflammation, activity and H. pylori density as histologically determined.

Conclusion Certain point mutations in H. pylori genome that affect susceptibility to clarithromycin correlate with histological features of gastritis.

Keywords Clarithromycin resistance, ClariRes assay, real-time PCR, quantitative real-time PCR, histology, Sydney classification, Helicobacter pylori infection