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Low Helicobacter pylori primary resistance to clarithromycin in gastric biopsy specimens from dyspeptic patients of a city in the interior of Sao Paulo, Brazil.

ABSTRACT: BACKGROUND: Clarithromycin, amoxicillin, and a pump proton inhibitor are the most common drugs recommended as first-line triple therapy for H.pylori treatment, which results in eradication rates close to 80%, varying regionally, principally due to emergency cases and increases of clarithromycin resistant strains. Nucleotide substitutions at the H. pylori domain V of the 23S rRNA fraction are involved in the macrolide resistance and the A2142G and A2143G mutations are predominant in clinical isolates worldwide including in Brazil. As H. pylori culture is fastidious, we investigated the primary occurrence of H. pylori A2142G and A2143G rDNA 23S mutations using a molecular approach directly on gastric biopsies of dyspeptic patients consecutively attended at Hospital das Clinicas of Marilia, São Paulo, Brazil. METHODS: Biopsy specimens obtained from 1137 dyspeptic patients, were subjected to histopathology and H. pylori diagnosis by histology and PCR. PCR/RFLP assay was used to detect A2142G and A2143G point mutations at domain V of the H. pylori 23S rDNA associated with clarithromycin resistance. Through the developed assay, a 768 bp PCR amplicon corresponding to1728 to 2495 bp of the 23S H. pylori rDNA is restricted with MboII for A2142G mutation detection and with BsaI for A2143G mutation detection. Occurrence of 23S rDNA A2142G results in two DNA fragments (418 and 350 bp) and of 23S rDNA A2143G results in three DNA fragments (108, 310 and 350pb), due to a conserved BsaI restriction site. RESULTS: The PCR method used to diagnose H. pylori presented sensitivity, specificity and accuracy of 77,6%, 79,3% and 78,6%, respectively, compared to histology, the gold standard method for H. pylori diagnosis used in our routine. Prevalence of H.pylori with clarithromycin resistant genotypes was 2,46%, with predominance of A2143G 23S rDNA point mutation. CONCLUSIONS: The PCR/RFLP assay was a rapid and accurate H.pylori diagnostic and clarithromycin resistance determination method useful for routine practice. As prevalence of primary resistance of H.pylori to clarithromycin due to A2142G and A2143G mutations remains low in Marilia, the standard clarithromycin containing triple therapy is still valid.

SUBMITTER: Suzuki RB

PROVIDER: S-EPMC4235177 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Low Helicobacter pylori primary resistance to clarithromycin in gastric biopsy specimens from dyspeptic patients of a city in the interior of São Paulo, Brazil.

Suzuki Rodrigo Buzinaro RB Lopes Rodrigo Augusto Basso RA da Câmara Lopes George Arouche GA Hung Ho Tin T Sperança Márcia Aparecida MA

BMC gastroenterology 20131204

<h4>Background</h4>Clarithromycin, amoxicillin, and a pump proton inhibitor are the most common drugs recommended as first-line triple therapy for H.pylori treatment, which results in eradication rates close to 80%, varying regionally, principally due to emergency cases and increases of clarithromycin resistant strains. Nucleotide substitutions at the H. pylori domain V of the 23S rRNA fraction are involved in the macrolide resistance and the A2142G and A2143G mutations are predominant in clinic ...[more]

PMID: 24305035

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Project description:BACKGROUND: Treatment effectiveness of Helicobacter pylori varies regionally and is decreasing worldwide, principally as a result of antibiotic resistant bacterium. Tetracycline is generally included in second line H. pylori eradication regimens. In Brazil, a high level of tetracycline resistance (TetR) is mainly associated with AGA926-928TTC 16?S rDNA nucleotide substitutions. As H. pylori culture is fastidious, we investigated the primary occurrence of H. pylori 16?S rDNA high level TetR genotype using a molecular approach directly on gastric biopsies of dyspeptic patients attending consecutively at Hospital das Clinicas of Marilia, São Paulo, Brazil. METHODS: Gastric biopsy specimens of 68 peptic ulcer disease (PUD) and 327 chronic gastritis (CG) patients with a positive histological diagnosis of H. pylori were investigated for TetR 16?S rDNA genotype through a molecular assay based on amplification of a 16?S rDNA 545?bp fragment by polymerase chain reaction and HinfI restriction fragment length polymorphism (PCR/RFLP). Through this assay, AGA926-928TTC 16?S rDNA TetR genotype resulted in a three DNA fragment restriction pattern (281, 227 and 37?bp) and its absence originated two DNA fragments (264 and 281?bp) due to a 16?S rDNA conserved Hinf I restriction site. RESULTS: The 545?bp 16?S rDNA PCR fragment was amplified from 90% of gastric biopsies from histological H. pylori positive patients. HinfI RFLP revealed absence of the AGA926-928TTC H. pylori genotype and PCR products of two patients showed absence of the conserved 16?S rDNA HinfI restriction site. BLASTN sequence analysis of four amplicons (two conserved and two with an unpredicted HinfI restriction pattern) revealed a 99% homology to H. pylori 16?S rDNA from African, North and South American bacterial isolates. A nucleotide substitution abolished the conserved HinfI restriction site in the two PCR fragments with unpredicted HinfI RFLP, resulting in an EcoRI restriction site. CONCLUSIONS: H. pylori AGA926-928TTC 16?S rDNA gene substitutions were not found in our population. More research is required to investigate if H. pylori TetR has a different genetic background in our region and if the nucleotide substitutions of the uncultured H. pylori 16?S rRNA partial sequences have biological significance.

Project description:A very simple, practical, sensitive, and specific colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA is described. This assay, which combines a sensitive sandwich DNA hybridization reaction and a colorimetric protocol similar to those used in conventional enzyme immunoassays, was shown to be suitable for detecting H. pylori-infected gastric biopsy specimens and for monitoring the eradication of the pathogen after treatment. The specificity and sensitivity of the colorimetric hybridization assay were tested by assaying 27 H. pylori strains (4 reference and 23 clinical isolates), 9 strains of other Helicobacter spp. or Campylobacter spp., and 11 clinical isolates of other urease-positive bacteria. The likelihood of H. pylori detection in gastric biopsy specimens by the colorimetric hybridization assay was evaluated with 23 H. pylori-positive and 41 H. pylori-negative biopsy specimens on the basis of positive and negative results, respectively, of culture, rapid urease test, histological examination, and PCR. Biopsy specimens from 33 treated patients, endoscopied 4 to 8 weeks after the end of treatment, were also tested. All H. pylori strains showed positive results in the colorimetric hybridization assay, presenting optical densities at 450 nm (OD450S) of > or = 3.0. None of the other Helicobacter spp., Campylobacter spp., or the clinical isolates of other urease-positive bacteria showed OD450S equal to or greater than the cutoff (mean OD450 cutoff, 0.208). The colorimetric hybridization assay detected all 23 H. pylori-positive biopsy specimens (mean OD450, 2.910 +/- 0.295), while none of the H. pylori-negative biopsy specimens was shown to be positive in the assay (mean OD450, 0.108 +/- 0.025). H. pylori was considered to be not eradicated from three of the posttreatment biopsy specimens by culture, rapid urease test, histological examination, and PCR. They were all positive by the colorimetric hybridization assay, and their OD450S were > or = 3.0. The colorimetric hybridization assay also detected two other H. pylori-positive patients. Specimens from these two patients had negative culture, rapid urease test, and histology results, and a specimen from one of them also tested negative by PCR. These results indicate that the colorimetric hybridization assay is a suitable method both for the diagnosis of H. pylori in biopsy specimens and for the follow-up of patients after the end of treatment.

Dataset Information

Low Helicobacter pylori primary resistance to clarithromycin in gastric biopsy specimens from dyspeptic patients of a city in the interior of Sao Paulo, Brazil.

Publications

Low Helicobacter pylori primary resistance to clarithromycin in gastric biopsy specimens from dyspeptic patients of a city in the interior of São Paulo, Brazil.

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