Abstract
BackgroundTriple therapy is the gold standard treatment for Helicobacter pylori eradication from the human stomach, but increased resistance to clarithromycin became the main factor of treatment failure. Until now, fastidious culturing methods are generally the method of choice to assess resistance status. In this study, a new genotypic method to detect clarithromycin resistance in clinical samples, based on fluorescent in situ hybridization (FISH) using a set of peptide nucleic acid probes (PNA), is proposed.ResultsThe set of probes targeting the point mutations responsible for clarithromycin resistance was applied to H. pylori suspensions and showed 100% sensitivity and specificity (95% CI, 79.9-100 and 95% CI, 71.6-100 respectively). This method can also be amenable for application to gastric biopsy samples, as resistance to clarithromycin was also detected when histological slides were tested.ConclusionsThe optimized PNA-FISH based diagnostic method to detect H. pylori clarithromycin resistance shown to be a very sensitive and specific method for the detection of clarithromycin resistance in the H. pylori smears and also proved to be a reliable method for the diagnosis of this pathogen in clinical samples and an alternative to existing plating methods.
Highlights
Triple therapy is the gold standard treatment for Helicobacter pylori eradication from the human stomach, but increased resistance to clarithromycin became the main factor of treatment failure
When peptide nucleic acid (PNA) probes are attached to a fluorochrome dye, they can be detected by epifluorescence microscopy or flow cytometry using the fluorescence in situ hybridization (FISH) method [16,17,20]
Some reports presented clarithromycin resistance mechanisms other than point mutations, such as efflux pumps or rRNA methylation [30] that can be revealed with phenotypic methods, they are not detected by genotypic methods that are specific to Genotype Polymerase chain reaction (PCR) E-test PNA-FISH
Summary
Triple therapy is the gold standard treatment for Helicobacter pylori eradication from the human stomach, but increased resistance to clarithromycin became the main factor of treatment failure. A new genotypic method to detect clarithromycin resistance in clinical samples, based on fluorescent in situ hybridization (FISH) using a set of peptide nucleic acid probes (PNA), is proposed. The antibiotic susceptibility has been detected in clinical laboratories by several phenotypical methods such as the agar dilution method, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) [6], or the alternative E-test that is considered to be more simple [7,8,9,10] These methods are fastidious, time consuming [11], and fail to give any information about the point mutation within the sample [3]. Due to the importance of antibiotic resistance, the aim of this work was to develop and validate a new PNA-FISH based diagnostic method to detect H. pylori clarithromycin resistance directly in paraffin embedded gastric biopsies
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