A2142G Point Mutations Research Articles

Antimicrobial resistance pattern of Helicobacter pylori in patients evaluated for dyspeptic symptoms in North-Eastern India with focus on detection of clarithromycin resistance conferring point mutations A2143G and A2142G within bacterial 23S rRNA gene

PurposeIn India there is evidence of antimicrobial resistance in Helicobacter pylori, a definitive pathobiont whose only known niche is human gastric mucosa. This in turn can lead to failure of treatment, persistence or chronicity of infection. This hospital based, prospective, observational study investigates the presence of antimicrobial resistance in the organism with focus on detection of A2143G and A2142G major point mutations in domain V of H. pylori 23S rRNA gene as a molecular mechanism of conferring resistance. MethodsEndoscopic gastric biopsy samples from 52 patients presenting with dyspeptic symptoms from January 2016 to December 2016 were subjected to culture in a microaerophilic environment using Campylobacter agar with for 2–5 days. Isolates were identified using gram-staining, motility test and biochemical reactions. Modified Kirby-Bauer Disc diffusion method was used to determine antimicrobial susceptibility against Clarithromycin, Metronidazole, Amoxycillin, Levofloxacin, Tetracycline, Cotrimoxazole and Erythromycin. Additionally, detection of A2143G and A2142G point mutations conferring Clarithromycin resistance was carried out using real time PCR following extraction and quantification of bacterial DNA. Histopathological examination was carried out on all biopsy samples. Descriptive and inferential statistical analytical methods were used. Differences were considered significant for p < 0.05. ResultsCulture positivity for H. pylori by phenotypic method was found to be 36.54%. Histopathologic Examination detected H. pylori in 55.7% and PCR detected 48.08% for either the wild type or one of two mutant strains A2143G and A2142G. No sample was found positive for both mutations. Metronidazole showed the highest resistance among antibiotics (78.9%) followed by Clarithromycin (47.3%). ConclusionPrevalence of antimicrobial resistance in H. pylori in North-Eastern India is substantially high with A2143G mutation being clinically most important in conferring Clarithromycin resistance. This resistance might be associated with low eradication rates despite initiation of therapy. ROC analysis of PCR proved it to be a good diagnostic tool.

Effectiveness of 7-day triple therapy with half-dose clarithromycin for the eradication of Helicobacter pylori without the A2143G and A2142G point mutations of the 23S rRNA gene in a high clarithromycin resistance area.

Tailored therapy has been widely used for patients with Helicobacter pylori (H. pylori) infection in South Korea. Herein, we evaluated the treatment outcomes of tailored clarithromycin-based triple therapy (TT) in patients infected with H. pylori. We enrolled 460 patients without A2142G and A2143G point mutations by dual priming oligonucleotide-based polymerase chain reaction who had taken TT and undergone the urease breath test to evaluate eradication in clinical practice. Eradication rates according to the treatment duration and dose of clarithromycin were analyzed. Among 460 patients (164 women, median age 63.0 years), 250 patients underwent TT with full-dose clarithromycin (TT-full CLA), and 216 patients underwent TT with half-dose clarithromycin (TT-half CLA). The eradication rates were 88.0% (220/250) in patients with TT-full CLA and 85.2% (179/210) in patients with TT-half CLA. In 250 patients with TT-full CLA, the eradication rates were 86.8% (33/38) in patients with 7-day TT-full CLA and 88.2% (187/212) in patients with 10-day or 14-day TT-full CLA (P = 0.788). In 210 patients with TT-half CLA, the eradication rates were 84.2% (139/165) in those with a 7-day TT-half CLA and 88.9% (40/45) in those with a 10-day or 14-day TT-half CLA (P = 0.436). For patients with H. pylori infection without A2142G and A2143G point mutations by DPO-PCR in clinical practice, treatment extension above 7-day TT with full CLA did not improve the eradication rates. Future studies on the treatment outcomes of TT-half CLA considering effectiveness and compliance are warranted.

Detection of A2143G, A2142C, and A2142G Point Mutations with Real-Time PCR in Stool Specimens from Children Infected with Helicobacter pylori.

Reports have indicated an increasing prevalence of clarithromycin resistance in children relative to adults. Thus, it is important to investigate primary clarithromycin resistance before therapy to avoid treatment failure. A2142G, A2143G, and A2142C point mutations in the peptidyltransferase region of the 23S ribosomal RNA (rRNA) of Helicobacter pylori (H. pylori) strains isolated from children with gastrointestinal symptoms and asymptomatic children were evaluated via real-time polymerase chain reaction (RT-PCR) using fecal DNA samples. The presence of H. pylori was determined using a fecal H. pylori antigen enzyme-linked immunosorbent assay (ELISA) kit from the stools of children (n = 543). A2143G, A2142C, and A2142G point mutations were detected via RT-PCR and confirmed by sequencing the 23S rDNA. Fecal H. pylori antigen testing was positive in 101 symptomatic (49) and asymptomatic (52) children. A significant difference was found between the 0–5- and 5–18-year-old groups in terms of the A2143G and A2142G point mutations (p = 0.001). The A2142C mutation was not detected. There was a significant difference in the A2143G mutation between the symptomatic and asymptomatic 5–18-year-old children (p = 0.019). Macrolides are frequently used to treat upper respiratory tract infections in children due to their selective pressure effect. We suggest that H. pylori strains carrying mutations in the 23S RNA subunit conferring clarithromycin resistance may lead to an intense inflammatory response in the gastric epithelial cells, allowing them to proliferate more rapidly and causing possible diarrhea, halitosis, or abdominal pain in children.

High prevalence of clarithromycin resistant Helicobacter pylori in Turkish children with gastric disorders

ObjectivesHelicobacter pylori (H. pylori) is one of the leading cause of most gastroduodenal diseases. Commonly, clarithromycin is prescribed to treat H. pylori infections. Increasing resistance to clarithromycin is now one of the main problems of health worldwide. This study is aimed to survey clarithromycin resistant H. pylori infection in children with gastric disorders in Turkey. Patients and methodsA total of 110 biopsy samples were collected from the gastric antral of children referred to Balcali Hospital in Adana city, Turkey from October 2015 to November 2016. After the DNA extraction, a PCR reaction was performed for the detection of glmM gene as a diagnostic marker for Helicobacter. To examine resistance to clarithromycin, RFLP-PCR was performed to detect A2142G and A2143G point mutations in the 23SrRNA amplification product. ResultsBased on histopathological and RUT tests and PCR, H. pylori was detected in 26.4% (29/110), 23.6% (26/110), and 33.6% (37/110) of the pediatric patients respectively. The frequency of H. pylori infection was dependent on age but independent of gender. The A2143G and A2142G point mutations were found in (n: 13, 61.90%) and (n: 8, 38.09%) of H. pylori isolates. ConclusionOur findings highlight a high rate of resistance to clarithromycin in Turkish children with H. pylori infection. Hence, it is essential to note other classes of antibiotics except clarithromycin for the effective treatment of H. pylori infection in children younger than 18 years in the study region.

A2143G and A2142G Point Mutations Within Bacterial 23S rRNA Gene in &lt;i&gt;Helicobacter pylori&lt;/i&gt; Confer Clarithromycin Resistance in Patients Evaluated for Dyspeptic Symptoms in North-Eastern India

A2143G and A2142G Point Mutations Within Bacterial 23S rRNA Gene in &lt;i&gt;Helicobacter pylori&lt;/i&gt; Confer Clarithromycin Resistance in Patients Evaluated for Dyspeptic Symptoms in North-Eastern India

Types of 23S Ribosomal RNA Point Mutations and Therapeutic Outcomes for Helicobacter pylori.

Background/AimsPoint mutations in the 23S ribosomal RNA gene have been associated with Helicobacter pylori clarithromycin resistance. This study aimed to detect the prevalence of these point mutations and to investigate the role of different point mutations in the success of eradication therapy.MethodsWe retrospectively investigated a total of 464 consecutive patients who underwent an endoscopic examination and dual-priming oligonucleotide-based multiplex polymerase chain reaction for H. pylori between June 2014 and October 2019. For 289 patients with negative point mutations, standard triple therapy was used in 287 patients, and the bismuth-quadruple regimen was used in two patients. For 175 patients with positive point mutations (A2142G, A2143G, and both mutations), standard triple and bismuth-quadruple therapies were used in 37 patients and 138 patients, respectively.ResultsThe eradication rates of standard triple and bismuth-quadruple therapies showed no significant difference in mutation-negative patients or those with the A2142G point mutation. However, the eradication rate with bismuth-quadruple therapy was significantly higher than that with standard triple therapy in the group with the A2143G mutation or with the double mutation. The eradication rates for standard triple and bismuth-quadruple therapies, respectively, were 25.8% and 92.1% in the per-protocol group (p<0.001) and 24.2% and 85.2% in the intention-to-treat analysis (p<0.001).ConclusionsThe A2143G point mutation is the most prevalent cause of clarithromycin resistance. Bismuth-quadruple therapy is superior to standard triple therapy in patients with the A2143G or double point mutation.

Usefulness of rapid urease test samples for molecular analysis of clarithromycin resistance in Helicobacter pylori

Helicobacter pylori is a gastric pathogen that is widely recognized as a causative agent of gastric disease. Its eradication is variable, mainly due to increased resistance to clarithromycin. Our objective was: to evaluate (i) if the biopsy specimen used for the rapid urease test is a useful sample to detect resistance to clarithromycin by PCR-RFLP and (ii) the distribution of A2142G and A2143G point mutations in the 23S rRNA gene, in relation to virulence factors in our region. Gastric specimens were collected from adult dyspeptic patients (n=141) and H. pylori was investigated by the rapid urease test, histopathological analysis and PCR for the hsp60 gene. Clarithromycin resistance was detected by PCR-RFLP in 62 H. pylori (+) paired biopsy specimens submitted to molecular analysis and the rapid urease test. H. pylori virulence factors were analyzed by multiplex PCR using specific primers for the cagA, vacA and babA2 genes. Thirteen out of 62 strains (20.9%) were resistant to clarithromycin: 6/13 (46.2%) harbored the A2143G mutation whereas 7/13 (53.8%) carried the A2142G point mutation. vacA m1s1 was the most frequent genotype among the resistant strains. In conclusion, the biopsy specimens used for the rapid urease test were suitable samples for clarithromycin resistance detection in patients infected with H. pylori, which became especially useful in cases where the number or size of the biopsies is limited. In addition, this is the first report of a molecular analysis for clarithromycin resistance performed directly from gastric biopsies in our region.

High rate of A2142G point mutation associated with clarithromycin resistance among Iranian Helicobacter pylori clinical isolates.

This study aimed to investigate the clarithromycin resistance and its associated molecular mechanisms among Helicobacter pylori isolates from dyspeptic patients in Shiraz, Iran. From January to May 2014, 100 H. pylori strains were isolated from patients with gastroduodenal disorders. The resistance to clarithromycin was quantitatively evaluated, using Epsilometer (E-test) method. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed on all the isolates to detect A2143G and A2142G mutations in 23S rRNA gene. The H. pylori isolation rate was found to be 31.4%. E-test showed that 20% of isolates were resistant to clarithromycin (MIC ≥ 1 mg/L). MIC of clarithromycin ranged between 0.016 and 24 mg/L. Findings of PCR-RFLP showed that the A2142G was the most (90%) frequently point mutation, followed by the A2143G (10%). No statistically significant difference was found between H. pylori clarithromycin resistance point mutations and patients' gender or age. To the best of our knowledge, this is the first report of high frequency of A2142G point mutation in Iran and probably in other regions of the world. Considering the increasing trend of H. pylori resistance to clarithromycin due to these mutations, it is crucial to investigate the new therapeutic approaches against H. pylori infection.

Helicobacter pylori infection: Correlation to disease severity and Clarithromycin resistance in a Sri Lankan setting

Background: Helicobacter pylori is a causative agent of gastritis, gastric ulcer, duodenal ulcer and gastric cancer. Clarithromycin is often used in the treatment of H. pylori infections in Sri Lanka. Although resistance of H. pylori to clarithromycin has been reported in other countries the situation in Sri Lanka is an enigma. We determined clarithromycin resistance of H. pylori by detecting two major point mutations (A2142G and A2143G) in the 23S rRNA gene. Further we assessed the histology of gastric mucosa of dyspeptic patients as a reasonably good predictor of cancer risk specially, in H. pylori positive patients. Methods & Materials: The study was a cross-sectional, descriptive study where 138 dyspeptic patients undergoing endoscopy examination were included. Ethical approval was granted from the ethical review committee, University of Sri Jayewardenepura (No-723). H. pylori infection was diagnosed by Polymerase chain reaction (PCR) amplification of the glmM gene of H. pylori. A2142G and A2143G point mutations associated with clarithromycin resistance were determined by PCR restriction fragment length polymorphism (RFLP). Histological features of the gastric mucosa were examined using H & E stain and gastritis was classified microscopically according to the updated Sydney system. Results: Seventeen percent (24/138) of the dyspeptic patients were positive for H. pylori by PCR. Of them 13 were males (54%) while 11 were females (46%). All H. pylori strains had a point mutation at A2142G, while A2143G mutation was not detected. Based on histological findings, 15 patients were diagnosed as H. pylori associated chronic active gastritis. Though mild to moderate infiltration of polymorphonuclear and mononuclear cells were observed in all H. pylori positive patients, gastric atrophy and metaplasia were not observed. Conclusion: This is the first report describing the presence of A2142G point mutation which is associated with clarithromycin resistance in a Sri Lankan population. It is therefore important to determine the eradication efficacy of H. pylori following clarithromycin treatment in Sri Lanka which can give an insight regarding the pheno-typical expression of the A2142G mutation. Further the proportion of H. pylori infections was found to be 17% in Sri Lanka.

Detection of clarithromycin-resistant Helicobacter pylori strains in a dyspeptic patient population in Sri Lanka by polymerase chain reaction-restriction fragment length polymorphism

Aim: The aim of this study was to investigate the proportion of common clarithromycin-resistant mutation types present in the 23S ribosomal ribonucleic acid (rRNA) gene of H. pylori strains in Sri Lanka. Settings and Design: The study was a cross-sectional, descriptive study where 76 dyspeptic patients who were required to undergo endoscopy examination were included. The study was carried out at a Teaching Hospital in Sri Lanka. Subjects and Methods: In-house urease test and polymerase chain reaction (PCR) amplification of the glmM gene of H. pylori was performed to confirm the H. pylori infection. Analysis of point mutations in 23S rRNA gene strains were performed by PCR-restriction fragment length polymorphism (RFLP). Results: Of the 16 urease-positive biopsies, 94% (n = 15) were positive by PCR using the glmM primer. All H. pylori strains yeilded a point mutation at A2142G site of the 23S rRNA gene, while A2143G mutation was not detected. Conclusions: For the first time in Sri Lanka, we reported predominance of A2142G point mutation associated with claritromycin resistance of H. pylori in a Sri Lankan population.

Detection of four clarithromycin resistance point mutations in Helicobacter pylori: comparison of real-time PCR and PCR-RFLP methods

Helicobacter pylori infection can be eradicated in up to 90 % of patients using current combination of triple therapies, of which clarithromycin is a key component. The development of clarithromycin resistance in H. pylori is recognized as a significant contributing factor in treatment failure. The aim of this study is to compare two fast and direct diagnostic methods to evaluate clarithromycin resistance in gastric biopsy specimens. This cross-sectional descriptive study was performed on 150 gastric biopsy specimens. Analysis for clarithromycin resistance was performed by real-time polymerase chain reaction (PCR) assay for A2144G, A2143G, A2143C, and A2142G point mutations by specific probes. Also, A2142G and A2143G point mutations were detected by PCR-RFLP method and using BsaI and MboII restriction enzymes. Out of 150 samples, 96 (64 %) clarithromycin-sensitive- and 54 (36 %) clarithromycin-resistant strains were detected by TaqMan real-time PCR assay. Thirty-seven (24.67 %) resistant strains were detected by PCR-RFLP method. Results showed that real-time PCR assay has enough accuracy to identify clarithromycin resistance and their mutations in a short time.