PCR detection of clarithromycin-susceptible and -resistant Helicobacter pylori from formalin-fixed, paraffin-embedded gastric biopsies

Abstract

Antimicrobial resistance to clarithromycin is a growing concern in the treatment of Helicobacter pylori and is associated with three major point mutations of the 23S rRNA, A2142C, A2142G, and A2143G. The use of traditional culture-based methods for determination of clarithromycin resistance in H. pylori are time consuming and lack sensitivity. We implemented a real-time PCR with melt curve analysis to detect and characterize H. pylori in formalin-fixed, paraffin-embedded gastric biopsy specimens to assess the frequency of clarithromycin resistance mutations in our study population. One hundred and fifty-three formalin-fixed, paraffin-embedded gastric biopsies were chosen on the basis of positive immunohistochemical staining for H. pylori and an accompanying histopathological diagnosis of Helicobacter-associated gastritis. New adjacent sections were taken for immunohistochemical staining and DNA extraction with subsequent testing by PCR assay and melt curve analysis using a primer and probe combination first described by Oleastro et al. 12 One hundred and forty-six samples demonstrated adequate amplification of a human DNA control target. Of these, there were 122 H. pylori immunohistochemistry-positive samples. In all, 103 out of 122 (84%) immunohistochemistry-positive samples demonstrated amplifiable H. pylori 23S rRNA gene target and 19 (16%) demonstrated no amplification of H. pylori. Twenty-two samples were negative for H. pylori by immunohistochemistry and PCR. Two were negative for H. pylori by immunohistochemistry, but were positive for H. pylori by PCR. In all, 52 out of 105 (50%) PCR-positive samples demonstrated resistance mutations, and it was determined that a heterogeneous population of mutated and unmutated organisms was present in 11 out of 52 samples. The use of PCR assays allows for a timely assessment of clarithromycin resistance status without the disadvantages of culture-based methods, and may lead to a decrease in treatment failure rates.

Clarithromycin resistance and 23S rRNA gene point mutations of Helicobacter pylori infection in children

“…In our study, mutation A2142C was not found in any patient, A2143G and A2142G mutations were found to be the most frequent. There are several other studies whose results are consistent with our study 20,28,[29][30][31] . The eradication ratio was lower for patients detected to have A2143G mutation 29 .…”

“…It is an advantage of this method to be able to determine the possible effect of clarithromycin resistance in the failure of treatment through the biopsy samples of previously applied treatments retrospectively. It is reported that concerning the formalin-fixed, paraffin-embedded gastric biopsies, the sensitivity of the PCR method can be enhanced through optimal control of time of fixation by formalin and proper choice of paraffin sections 28 . PCR method is particularly more effective in identifying H.pylori infection for cases having less number of bacteria 28 .…”

“…It is reported that concerning the formalin-fixed, paraffin-embedded gastric biopsies, the sensitivity of the PCR method can be enhanced through optimal control of time of fixation by formalin and proper choice of paraffin sections 28 . PCR method is particularly more effective in identifying H.pylori infection for cases having less number of bacteria 28 . In our study, mutation A2142C was not found in any patient, A2143G and A2142G mutations were found to be the most frequent.…”

In situ targeted MRI detection of Helicobacter pylori with stable magnetic graphitic nanocapsules

“…Biopsy-based testing is commonly performed to detect early H. pylori infection34. This panel of tests includes culture5, urease test6, histology7 and PCR8, all of which provide direct and accurate information. However, biopsy is an invasive and tedious procedure, requiring endoscopy that is prone to missing small lesions and making the patient uncomfortable.…”

Helicobacter pylori infection

“…Several PCR-based methods have been developed for rapid detection of antibiotic resistant H. pylori strains [15,16]. Several PCR-based methods have been developed for rapid detection of antibiotic resistant H. pylori strains [15,16].…”