Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy specimens

Abstract

A biprobe real-time PCR protocol, followed by hybridization melting point analysis, to detect point mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance was established and evaluated in a clinical study. Of 92 patients who underwent endoscopy, 45 were found to be H. pylori infected and invariably were also culture positive. Of the 45 isolates, 11 were shown to be resistant to clarithromycin by E-test. With respect to the detection of H. pylori infection, PCR showed sensitivities of 100% in biopsies and 98% in stool specimens and a specificity of 98% in both biopsy and stool samples. All clarithromycin-sensitive cases were identified as such by PCR in both biopsy and stool samples. Of the cases with a resistant strain, eight were identified as such in stool DNA and nine were identified in biopsy DNA. Failure of PCR to detect the resistant genotype in the biopsy DNA, stool DNA, or both (one case) was associated with mixed populations. In these cases, patients had not been treated for H. pylori infection before, and the sensitive population showed to be present in considerably higher numbers than the resistant population. In five of six cases in which infection with a resistant genotype only was identified by PCR, the patients had received clarithromycin-based eradication therapy in the past. Thus, the assay presented provides a highly accurate noninvasive method to detect H. pylori infection in stool and at the same time allows for culture-independent clarithromycin susceptibility testing.

FIG. 1.

(A and B) Comparison of Cp values obtained by the ureA assay to those obtained by the 23S rRNA gene assay for biopsies (A) and stool specimens (B). (C and D) Comparisons of Cp values between biopsy and stool samples for the ureA assay (C) and the 23S rRNA gene assay (D); three cases with positive biopsy but negative stool PCR results are not shown.

FIG. 2.

Melting peaks obtained from stool DNA extracts of patients infected with a clarithromycin-sensitive (wild-type) and/or a clarithromycin-resistant (mutant) genotype. Sensitive genotypes showed a melting temperature of 63°C. Resistant genotypes with an A-to-G transition showed a melting temperature of 54°C. Mutants with an A-to-C transversion showed a melting temperature of 58°C (the curve was obtained from purified H. pylori DNA, since no such mutation was detected in the stool samples of patients). No biprobe-specific melting curves were obtained from noninfected patients. Values on the y axis are the first negative derivative of the change in fluorescence (dF) divided by the change in temperature (dT) (i.e., −dF/dT).