PCR-RFLP DETECTION OF POINT MUTATIONS CONFERRING RESISTANCE TO CLARITHROMYCIN OF HELICOBACTER PYLORI IN QUANG NGAI

2015 ◽  
pp. 54-61
Author(s):  
Ngoc Doanh Pham ◽  
Van Huy Tran ◽  
Thi Minh Thi Ha
Keyword(s):  
Helicobacter Pylori ◽  
General Hospital ◽  
Chronic Gastritis ◽  
Point Mutations ◽  
23S Rrna ◽  
Rrna Gene ◽  
Clarithromycin Resistance ◽  
Urease Test ◽  
Pcr Rflp ◽  
H Pylori

Background: Clarithromycin resistance of H-pylori is the main cause leading to treatment failure. Aim: The purpose of this study was to determine the rate of clarithromycin resistance mutation on gene 23S ribosomal popular robonucleotide acid (rRNA) of H-pylori in patients with chronic gastritis in Quang Ngai General Hospital PCR-RFLP. Method: This is a cross-sectional study in 64 patients infected with H-pylori was determined by 3 methods and chronic gastritis proven by histology. Sample collection conducted in Quang Ngai general hospital and molecular biology tests were conducted in the medical genetics department Hue of Medical and Pharmaceutical University. Urease test, histopathological examination and perform HE staining PCR 23S rRNA gene fragment of H-pylori to determine H-pylori infection. Analysis of genetic mutations in the 23S rRNA point is performed by PCR-RFLP technique. Results: Of the 64 biopsies qualify included in the study, 41 samples with clarithromycin resistance point mutations (64%), of which 40 (62.5%) had mutations A2143A, one sample with A2142A (2%). No samples had mutations A2142C and no more than one mutation.Conclusion: This is the first time we report mutations related to clarithromycin of H-pylori in Quang Ngai province. Mutations rate is high (64%), among the common mutations, the most common mutantation is A2143G. Key words: Helicobacter pylori, clarithromycin resistance, PCR-RFLP, point mutations

STUDY ON POINT MUTATIONS AT POSITIONS 2132 AND 2143 IN 23S RRNA GENE OF HELICOBACTER PYLORI AMONG PATIENTS WITH CHRONIC GASTRITIS

2016 ◽  
pp. 12-20
Author(s):  
Thi Minh Thi Ha ◽  
Van Huy Tran ◽  
Viet Nhan Nguyen ◽  
Thanh Hoa Nguyen ◽  
Phan Tuong Quynh Le
Keyword(s):  
Helicobacter Pylori ◽  
Chronic Gastritis ◽  
Point Mutations ◽  
23S Rrna ◽  
Rrna Gene ◽  
Clarithromycin Resistance ◽  
Pcr Rflp ◽  
23S Rrna Gene ◽  
History Of ◽  
H Pylori

Background: Clarithromycin resistance in Helicobacter pylori has been found to be associated with point mutations at positions 2142 and 2143 in 23SrRNA gene. The aims of this study were: (1) to determine the rates of point mutations A2143G, A2142G and A2142C in 23SrRNA gene of H. pylori among patients with chronic gastritis by PCR-RFLP technique; and (2) to assessthe association between these mutations and some clinical, endoscopic and histopathological characteristics of chronic gastritis. Patients and methods: two hundreds and twenty six patients with H. pylori-positive chronic gastritis were determined A2143G, A2142G and A2142C mutations by PCR-RFLP technique with DNA extracted from endoscopic biopsy specimens of gastric mucosa. Results: The rate of point mutations at positions 2142 and 2143 in 23S rRNA gene of H. pylori was 35.4% in total, the A2143G and A2142G mutationsaccounted for 92.5% and 7.5% of all point mutations, respectively. No A2142C mutation was found. These mutations were not associated with age, gender,distribution of gastritis, and the presence of atrophic gastritis. The rate of A2143G mutation in groups with and without a history of clarithromycin treatment were 44.9% and 24.8%, respectively (p = 0,0065). The A2142G mutation was associated with intestinal metaplasia and/or dysplasia. Conclusion: The point mutations at positions 2142 and 2143 in 23S rRNA gene were found at a high rate in H. pylori strains amongpatients with chronic gastritis, with the absolute predominance of A2143G mutation. The A2143G mutation was associated with a history of clarithromycin treatment. Key words: 23S rRNA gene, Helicobacter pylori, A2143G, A2142G, A2142C mutation, clarithromycin resistance, chronic gastritis.

scholarly journals PCR-RFLP detection of point mutations A2143G and A2142G in 23S rRNA gene conferring resistance to clarithromycin in Helicobacter pylori strains.

2014 ◽  
Vol 61 (2) ◽  
Author(s):  
Karolina Klesiewicz ◽  
Paweł Nowak ◽  
Elżbieta Karczewska ◽  
Iwona Skiba ◽  
Izabela Wojtas-Bonior ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Point Mutations ◽  
Rflp Analysis ◽  
23S Rrna ◽  
Rrna Gene ◽  
Efficient Management ◽  
Clarithromycin Resistance ◽  
Pcr Rflp ◽  
23S Rrna Gene ◽  
H Pylori

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 were negative for both mutations. The average MIC values for A2143G and A2142G mutants were 6 and 30 mg/L, respectively. Frequencies of A2143G and A2142G mutations were the same in all isolates tested. Strains with A2143G mutation exhibited lower MIC values than A2142G mutants. Application of PCR-RFLP method for detection of clarithromycin resistance allows for better and more efficient management of H. pylori infections.

scholarly journals Helicobacter pylori 23S rRNA gene mutations associated with clarithromycin resistance in chronic gastritis in Vietnam

2018 ◽  
Vol 12 (07) ◽  
pp. 526-532 ◽  
Author(s):  
Van Huy Tran ◽  
Thi Minh Thi Ha ◽  
Phan Tuong Quynh Le ◽  
Viet Nhan Nguyen ◽  
Trung Nam Phan ◽  
...  
Keyword(s):  
Chronic Gastritis ◽  
23S Rrna ◽  
Rapid Urease Test ◽  
Rrna Gene ◽  
Wild Type ◽  
Clarithromycin Resistance ◽  
Urease Test ◽  
Pcr Rflp ◽  
23S Rrna Gene ◽  
H Pylori

Introduction: Data about the prevalence of the A2142C, A2142G, and A2143G mutations in 23S rRNA gene is still limited. The aim of this study was to determine the prevalence of these mutations in 23S rRNA gene of H. pylori vietnamese strains. Methodology: One hundred and sixty-nine patients with H. pylori-positive chronic gastritis were examined. H. pylori was detected by rapid urease test and Polymerase chain reaction (PCR). Total DNA was extracted from gastric biopsy specimens. A2142C, A2142G, and A2143G mutations were detected by DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP). Results: A2143G mutation was detected in 36.1% of samples, A2142G mutation in 3.6%, while A2142C mutation was not found in any case. The mixture of wild-type and mutation strains was found in 50% of specimens with A2142G, in 23% of specimens with A2143G mutation. There was no association of 23S rRNA gene point mutations with gender or age. However, an association between the heterogeneity of mutation and age was evidenced, with mean age of the group of pure A2143G higher than the group of wild-type/A2143G mixture, and rate of the wild-type/A2143G mixture higher in patients under 40 years of age. Conclusion: A2143G mutation was prominent, while A2142C mutation was not found in the 23S rRNA gene. PCR-RFLP has revealed a reliable assay allowing a rapid and cost-effective detection of clarithromycin-resistant strains. This is useful in countries as Vietnam with high prevalence of clarithromycin-resistance before choosing optimal therapy for H. pylori eradication.

scholarly journals Molecular Assessment of Resistance to Clarithromycin in Helicobacter pylori Strains Isolated from Patients with Dyspepsia by Fluorescent In Situ Hybridization in the Center of Iran

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Jina Vazirzadeh ◽  
Jamal Falahi ◽  
Sharareh Moghim ◽  
Tahmineh Narimani ◽  
Rahmatollah Rafiei ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
In Situ Hybridization ◽  
Point Mutations ◽  
23S Rrna ◽  
Rrna Gene ◽  
Clarithromycin Resistance ◽  
Genotypic Analysis ◽  
23S Rrna Gene ◽  
H Pylori

Background and Aims. Helicobacter pylori is a common infectious bacterium mostly found in gastroduodenal diseases. The increased prevalence of clarithromycin-resistant H. pylori strains is a major challenge in the successful treatment of infections caused by this organism. The present study is aimed at detecting the clarithromycin resistance pattern of H. pylori strains isolated from gastric biopsies and evaluating point mutations of the 23S rRNA gene. Patients and methods. In the present descriptive cross-sectional study, 165 patients with gastrointestinal disorders, who were referred to the Endoscopy Center of Dr. Shariati Hospital of Isfahan, Iran, were enrolled from April to July 2018. H. pylori infection was diagnosed by culture, and susceptibility of the isolates to clarithromycin was assessed by the E-test. Minimum inhibitory concentration (MIC) values were obtained based on EUCAST recommendations. Also, fluorescence in situ hybridization (FISH) was used to determine point mutations associated with clarithromycin resistance. Results. By using culturing, H. pylori was isolated from 50.3% (83/165) gastric biopsy specimens. The overall frequency of resistance to clarithromycin was 25.3% (21/83) by the E-test. In the resistance genotypic analysis, 19 isolates had mutations. The prevalence of A2143G and A2144G mutations was 68.4% (13/19) and 31.5% (6/19), respectively. A2143C mutation was not tracked in any isolate. Two isolates with MIC>0.5 μg/mL had no mutations that could be related to other mechanisms of resistance. Conclusion. As presented in the study, the high prevalence of clarithromycin-resistant H. pylori due to point mutations of the 23S rRNA gene indicates the necessity of revising the standard treatment regimen based on antibiotic susceptibility pattern of each region.

DETECTING POINT MUTATIONS A2142G AND A2143G IN THE 23S RRNA GENE CAUSING HELICOBACTER PYLORI RESISTANCE TO CLARITHROMYCIN BY PCR RFLP

2013 ◽  
pp. 56-63
Author(s):  
Thi Minh Thi Ha ◽  
Van Huy Tran
Keyword(s):  
Helicobacter Pylori ◽  
Point Mutations ◽  
23S Rrna ◽  
Rapid Urease Test ◽  
Rrna Gene ◽  
Peptic Ulcers ◽  
Urease Test ◽  
Pcr Rflp ◽  
Pcr Products ◽  
23S Rrna Gene

Background: Clarithromycine-resistance of H.pylori is one important cause of decreasing eradication rate of H.pylori. This study is aimed at: (1): evaluating the application of PCR RFLP in detecting point mutations A2142G and A 2143G in the 23SrRNA gene resulting Clarithromycine-resistant H.pylori. (2) determining the rate of these point mutations by PCR RFLP in patients with gastroduodenitis or peptic ulcers. Patients and methods: 38 patients with gastroduodenitis or peptic ulcer were enrolled. H.pylori infection was confirmed by rapid Urease test and PCR. PCR was performed in two phases: amplification of gene 23S rRNA containing A2143 and A2142, followed by digestion of PCR products by enzymes BbsI and BsaI in order to detecting point mutations A2143G and A2142G. Different quantities of enzyme of digestion were evaluated (5U; 7,5U; 10U; 15U and 20U) in order to determine an optimal quantity. Results: The components of digesting reaction were optimized as followings: performed in a solution volume of 20 µl, including 5 µl PCR products (to amplify gene 23S rRNA containing 2142 and 2143), 10 U enzyme (BsaI to detect A2143G, BbsI to detect A2142G), 2 µl buffer G 2X, and water for a sufficient volume. 17 point mutations A2143G were found (44.7%). Conclusion: PCR RFLP could be routinely applied to detect point mutations A2143G and A2142G in the 23S rRNA gene causing clarithromycine-resistant H.pylori. The rate of these point mutations in this group of patients was relatively high. Key words: Clarithromycin resistance, gene 23S rRNA, Helicobacter pylori

DETERMINATION OF MUTATIONS IN 23S RRNA GENE OF HELICOBACTER PYLORI AND THE ASSOCIATION OF THESE GENE MUTATIONS WITH CLARITHROMYCIN-RESISTANCE IN CHRONIC GASTRITIS

2015 ◽  
pp. 36-46
Author(s):  
Thi Minh Thi Ha ◽  
Van Huy Tran ◽  
Viet Nhan Nguyen ◽  
Phan Tuong Quynh Le ◽  
Trung Nam Phan ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Chronic Gastritis ◽  
Point Mutations ◽  
23S Rrna ◽  
Rrna Gene ◽  
Biopsy Specimens ◽  
Clarithromycin Resistance ◽  
Resistant Phenotype ◽  
23S Rrna Gene ◽  
Domain V

Background: Resistance of Helicobacter pylori to clarithromycin is mostly due to the point mutations in the 23S rRNA. The aims of the present study were to determine mutations in domain V of 23S rRNA gene of Helicobacter pylori in chronic gastritis patients by sequencing; and to assess the association of these mutations with clarithromycin-resistant phenotype determined by E-test. Patients and methods: Gastric mucosal biopsy specimens from 170 patients with chronic gastritis were entered in the study. DNA was extracted from biopsy specimens obtained by endoscopy and prepared for sequencing domain V of gene 23S rRNA. 80 biopsy specimens were determined MIC by E-test. Results: Eight point mutations were detected. At 2142 and 2143 sites of gene, A2143G and A2142G mutations were detected in 35.3% and 3.5%, respectively. Six remaining mutations were G2172T (0.6%), T2182C (86.5%), C2195T (5.3%), A2223G (42.9%), T2244C (98.2%) và A2302G (8.8%). A2143G mutation was associated with clarithromycin-resistant phenotype, OR = 368.58 (95%CI: 34.53 – 3934.12). The rate of clarithromycin-resistance in the group with A2143G mutation was 96.7%, while in the group without A2143G mutation was 10%. Predicted probabilities for clarithromycin-resistance were calculated by logistic regression model with the accuracy rate of 96.1%. Conclusions: There was the association between A2143G mutation and clarithromycin-resistant phenotype. Genotypes determined by sequencing domain V of 23S rRNA gene could be used to predict the probabilities of clarithromycin-resistance. Key words: 23S rRNA gene, clarithromycin-resistance, Helicobacter pylori

scholarly journals Rapid screening of clarithromycin resistance in Helicobacter pylori by pyrosequencing

2007 ◽  
Vol 56 (10) ◽  
pp. 1370-1376 ◽  
Author(s):  
Karen-Anja Moder ◽  
Franziska Layer ◽  
Wolfgang König ◽  
Brigitte König
Keyword(s):  
Helicobacter Pylori ◽  
Point Mutations ◽  
Rapid Screening ◽  
23S Rrna ◽  
Resistance Testing ◽  
Resistance Mutations ◽  
Additional Time ◽  
Clarithromycin Resistance ◽  
H Pylori ◽  
Domain V

Helicobacter pylori infections can be effectively treated with clarithromycin, a macrolide, in combination with other antibiotics, such as amoxicillin, tetracycline or metronidazole. The failure of H. pylori eradication is mainly associated with macrolide-resistant strains. Three point mutations (A2142G/C, A2143G, T2182C) in the peptidyltransferase region of domain V of the 23S rRNA have been described as being associated with clarithromycin resistance. Therefore, the determination of clarithromycin resistance by pyrosequencing was evaluated. H. pylori from 81 gastric biopsies was cultured and clarithromycin resistance was determined by Etest, as well as by pyrosequencing technology (PSQ 96 system; Biotage). The respective mutations were set in relation to the MIC measured in μg ml−1 by Etest. In this study, point mutations in positions 2142 and 2143 were associated with clarithromycin resistance. Mutations in position 2182 did not contribute to clarithromycin resistance. In addition, from 22 out of the 81 biopsies, clarithromycin resistance was determined directly without culturing H. pylori to save additional time. Identical results were obtained as compared to resistance testing with pure H. pylori strains. All results obtained by pyrosequencing were evaluated by Sanger sequencing. The data show that pyrosequencing to detect point mutation is a fast and reliable method for determining clarithromycin resistance in H. pylori, and provides the same results as the Etest.

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