Change of point mutations in Helicobacter pylori rRNA associated with clarithromycin resistance in Italy (original) (raw)
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Comparative Clinical Pathology, 2012
Currently, a seven-day, triple-drug regimen has been recommended as one of the first-line therapies for Helicobacter pylori management in which clarithromycin is a key component. Development of clarithromycin resistance leads to the long term assessment of the efficacy of clarithromycin in the triple-drug regimen. The aim of this study was to rapidly and directly assess clarithromycin resistance point mutations on gastric biopsy specimens by using PCR-RFLP method. Biopsy samples were obtained over a 6-months period of 2009, from 200 dyspeptic patients referred to Shahrekord University of Medical Sciences, Iran. Initially, rapid urease test was performed and then DNA was isolated from each tissue and used for molecular analysis such as PCR (for H. pylori diagnostic) and PCR-RFLP (for Cla resistance determination). RUT and PCR results showed that 164 (82%) of the patients were H. pylori-positive. Resistance was evaluated in 164 samples by using enzymes BsaI and MboII. Thirty nine (39) (23/78%) clarithromycin-resistant strains were detected which were identified as 15 (9.15%) A2143G, 15 (9.15%) A2142G and 9 (5.49%) mix strains. The results showed that PCR-RFLP method had a high accuracy to detect A2142G and A2143G mutations associated with resistance to clarithromycin in the minimum possible time.
Acta Biochimica Polonica, 2014
The occurrence of clarithromycin resistance among Helicobacter pylori strains is a major cause of the treatment failure. Resistance to this drug is conferred by point mutations in 23S rRNA gene and the most prevalent mutations are A2143G and A2142G. The aim of the study was to evaluate the occurrence of A2143G and A2142G mutations in a group of H. pylori strains resistant to clarithromycin. The study included 21 clarithromycin-resistant H. pylori strains collected between 2006 and 2009 in southern Poland. Resistance to clarithromycin was quantitatively tested with the E-test to determine the minimal inhibitory concentration (MIC value). The point mutations of H. pylori isolates were detected by PCR followed by RFLP analysis. The MIC values for clarithromycin for the analyzed strains ranged from 1.5 mg/L to 64 mg/L. Nine H. pylori strains exhibited A2143G mutation and A2142G mutation was found in 9 isolates as well. The results of RFLP analysis of 3 clarithromycin-resistant strains w...
Journal of Clinical Microbiology, 2003
The main cause of failure of Helicobacter pylori eradication therapy is resistance to clarithromycin. The resistance is due to three point mutations in two positions on the 23S rRNA (A2142C, A2142G, and A2143G). Our aim was to develop a rapid and accurate method to detect these mutations directly on biopsy specimens. We developed a real-time PCR that included a simultaneous detection of the amplicons by hybridization of two probes labeled with LC-Red and fluorescein by using the fluorescence resonance energy transfer (FRET) technology and melting curve analysis with the LightCycler thermocycler. The assay was first applied successfully on reference strains, reference plasmids, and H. pylori-negative biopsies. Biopsies from 200 patients having failed a first eradication attempt and for whom the H. pylori strain was available were then tested with the new assay. A result was obtained in 199 cases; a single genotype was detected in 157 cases, two genotypes were detected in 41 cases, and three genotypes were detected in one case. There were, in total, seven discrepancies between the real-time PCR and the phenotypic method of determination of clarithromycin susceptibility, and in an additional four cases the two phenotypic methods were in disagreement. PCR-restriction fragment length polymorphism was applied to a sampling of biopsies, including all of the cases with multiple genotypes and all the cases with discrepant results. Finally, in four cases with discrepant results, the real-time PCR detected the resistant population at a concentration so low that it could not be detected by the phenotypic method, while in three cases other mutations could be involved. This assay had an accuracy at least as satisfactory as that of the phenotypic tests and could be performed within 2 h, allowing it to be used before the administration of therapy in the case of a first H. pylori eradication.
Helicobacter, 2007
Background: The aim of our study was to assess the different mutations involved in clarithromycin-resistant Helicobacter pylori strains isolated from French children and their temporal trends. Methods: The point mutations of H. pylori were detected by PCR followed by RFLP technique in 50 clarithromycin-resistant strains collected between 1993 and 2004 in France. Results: Clarithromycin resistance was observed in 23% (50/217) of H. pylori isolates. Two mutations A2143G and A2142G in the 23S rRNA genes of H. pylori were detected. The former was found in 45/50 (90%) of isolates. The rate of resistance increased with time from 18.6% in the period 1993-1996 to 41.6% in 2001-2004. No significant difference was observed in the distribution of mutations during the same periods. No correlation was found between any mutation and age, sex, and ethnic origin of children. Furthermore, no significant differences in minimal inhibitory concentrations level were observed according to the different point mutations. In all cases, only one point mutation was present, except in two cases where two different mutations were found in two different clones from the same biopsy. Conclusion: The mutation A2143G is predominant in clarithromycin-resistant H. pylori strains isolated from children in France. We report for the first time the presence of two clarithromycin-resistant clones harboring two different mutations of the 23S rRNA genes present in the same biopsy specimen and genotypically identical as demonstrated by RAPD fingerprinting.
Alimentary Pharmacology and Therapeutics, 2006
Background Helicobacter pylori clarithromycin resistance is increasing worldwide and different mutations are involved in its mechanisms. Recently, molecular methods have been proposed to assess these mutations. Aim To assess prevalence of primary clarithromycin resistance in two Italian areas, and the distribution of involved mutations, by using a novel method for real-time polymerase chain reaction. Methods Two hundred and thirty-two H. pylori-positive patients undergoing oesophagogastroduodenoscopy in two Italian towns (Rome, centre Italy; Foggia, south Italy) were enrolled. Helicobacter pylori infection was detected by histology, rapid urease and urea breath tests. Clarithromycin resistance was assessed by TaqMan real-time polymerase chain reaction on paraffin-embedded antral biopsies. Results Primary clarithromycin resistance was detected in 62 (26.7%) patients. Its prevalence did not differ between the two areas (31.5%, centre vs. 23.3%, south; P ¼ 0.17) and between non-ulcer dyspepsia and peptic ulcer patients (28.4% vs. 20.7%, P ¼ 0.2). The A2143G point mutation was detected in 35 (56.4%) patients, A2142G in 14 (22.6%), A2142C in eight (12.9%), whilst a double mutation (A2143G plus A2142C or A2142G) was present in the remaining five (8.1%) cases. Conclusions Our study found that primary clarithromycin resistance is highly prevalent in both central and southern Italy, and that A2143G is the most frequent point mutation involved in these areas.
Clarithromycin Resistance and 23S rRNA Mutations in Helicobacter pylori
Gastrointestinal Endoscopy, 2011
To determine the 23S rRNA point mutations in clarithromycin resistance of Helicobacter pylori strains isolated from southwest, Iran. This was a cross-sectional survey, which was done on 263 patients who referred to endoscopy department of Shehrekord university of medical sciences. According to gram stain, urease, catalase, oxidase and polymerase chain reaction (PCR) H. pylori identified. Standard National Committee for Clinical Laboratory Standard (NCCLS) method used for assessment of clarithromycin resistance. Specific primers and restriction enzymes BsaI and MboII by PCR-RFLP were used for analysis of A2143G and A2142G mutations. So for the detection of A2142C, specific primers and PCR method were used. 84 strains of H. pylori (31.94%) determined by PCR method. Of 19 (22.62%) clarithromycin resistant strains 13 (68.40%), 3 (15.78%), 2 (10.52%) had A2143G, A2142G, A2142C respectively and one unknown mutation in 23S rRNA gene. Because of considerable resistance to clarithromycin, direct diagnosis of this mutation by molecular approach in other parts of the country is necessary.
Journal of Gastrointestinal and Liver Diseases Jgld, 2011
Background. Primary clarithromycin resistance markedly reduces Helicobacter pylori eradication rate following standard therapies. Prevalence of primary clarithromycin resistance in H. pylori is increasing, and three point mutations are mainly involved. Aim. To assess both the prevalence of primary clarithromycin resistance in Italy, and the distribution of the involved point mutations. methods. Primary clarithromycin resistance was assessed by TaqMan real-time polymerase chain reaction on antral biopsies of 253 consecutive, H. pylori infected patients enrolled in 13 Italian centres between January and September 2010. Results. Primary clarithromycin resistance was detected in 25 (9.9%) patients, with prevalence values widely ranging from 0 to 25%. Clarithromycin resistance rate was higher in female as compared to male patients (13.4% vs. 5.3%, p=0.03), and it tended to be higher in non-ulcer dyspepsia than in peptic ulcer patients (10.6% vs. 6.9%, p=0.5), female patients with non-ulcer dyspepsia showing the highest value (15.4%). The A2143G point mutation was detected in 13 (52.0%) patients, the A2142G in 9 (34.6%), whilst a double point mutation (A2143G plus A2142G) in 3 (11.6%) cases. Conclusions. Primary clarithromycin resistance is highly variable in different Italian geographic areas. High resistance rates were observed in female and in dyspeptic patients. Among
Microbes and Infectious Diseases, 2022
Background: Helicobacter pylori (H. pylori) colonizes the stomach and affect almost 50% of the world's population. Clarithromycin is considered a cornerstone for H. pylori treatment. Emergence of clarithromycin resistance (CLR-R) has played a major role in failure of H. pylori eradication both in adults and children. Clarithromycin resistance is mostly due to mutations in 23S rRNA gene: A2142G, A2142C, and A2143G. The aim of the current study is to determine the prevalence of CLR-R among H. pylori infected children with prior clarithromycin treatment. Materials and Methods: Multiple endoscopic gastric biopsies were collected from 50 H. pylori infected children after cessation of clarithromycin-based treatment. Samples were subjected to histopathological examinations, rapid urease test (RUT) and simultaneous molecular detection of H. pylori infection as well as CLR-R by multiplex Real-Time polymerase chain reaction (PCR). Results: Histopathological examinations and RUT revealed H. pylori in 74% and 92% of samples respectively. Molecular detection of CLR-R showed that 62.5% positive H. pylori cases were not harboring any of the tested mutations, while 25% harbored 2143A-G single mutation. Double mutations (2142A-C and 2143A-G) were detected in only 4 cases. Statistical significant correlation existed between both RUT and PCR results as well as between histopathological findings and PCR test results. Conclusions: A combination of histopathogy, RUT and multiplex PCR procedures offers a real benefit in the simultaneous diagnosis of H. pylori infection along with clarithromycin resistance status. Other mechanisms of clarithromycin resistance need to be investigated to explain treatment failure in absence of the previously detected mutations.