Change of point mutations in Helicobacter pylori rRNA associated with clarithromycin resistance in Italy

2014 ◽  
Vol 63 (3) ◽  
pp. 453-457 ◽  
Author(s):  
Vincenzo De Francesco ◽  
Angelo Zullo ◽  
Floriana Giorgio ◽  
Ilaria Saracino ◽  
Cristina Zaccaro ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Real Time ◽  
Real Time Pcr ◽  
Single Point ◽  
Point Mutations ◽  
Clarithromycin Resistance ◽  
Genotypic Analysis ◽  
Previously Treated ◽  
Resistant Strains ◽  
H Pylori

Primary clarithromycin resistance is the main factor affecting the efficacy of Helicobacter pylori therapy. This study aimed: (i) to assess the concordance between phenotypic (culture) and genotypic (real-time PCR) tests in resistant strains; (ii) to search, in the case of disagreement between the methods, for point mutations other than those reported as the most frequent in Europe; and (iii) to compare the MICs associated with the single point mutations. In order to perform real-time PCR, we retrieved biopsies from patients in whom H. pylori infection was successful diagnosed by bacterial culture and clarithromycin resistance was assessed using the Etest. Only patients who had never been previously treated, and with H. pylori strains that were either resistant exclusively to clarithromycin or without any resistance, were included. Biopsies from 82 infected patients were analysed, including 42 strains that were clarithromycin resistant and 40 that were clarithromycin susceptible on culture. On genotypic analysis, at least one of the three most frequently reported point mutations (A2142C, A2142G and A2143G) was detected in only 23 cases (54.8 %), with a concordance between the two methods of 0.67. Novel point mutations (A2115G, G2141A and A2144T) were detected in a further 14 out of 19 discordant cases, increasing the resistance detection rate of PCR to 88 % (P<0.001; odds ratio 6.1, 95 % confidence interval 2−18.6) and the concordance to 0.81. No significant differences in MIC values among different point mutations were observed. This study suggests that: (i) the prevalence of the usually reported point mutations may be decreasing, with a concomitant emergence of new mutations; (ii) PCR-based methods should search for at least six point mutations to achieve good accuracy in detecting clarithromycin resistance; and (iii) none of the tested point mutations is associated with significantly higher MIC values than the others.

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.

scholarly journals Clinical Evaluation of a Real-Time PCR Assay for Simultaneous Detection of Helicobacter pylori and Genotypic Markers of Clarithromycin Resistance Directly from Stool

2021 ◽  
Author(s):  
Rebecca Marrero Rolon ◽  
Scott A Cunningham ◽  
Jayawant N Mandrekar ◽  
Erin T Polo ◽  
Robin Patel
Keyword(s):  
Helicobacter Pylori ◽  
Real Time ◽  
Triple Therapy ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Pcr Assay ◽  
Clarithromycin Resistance ◽  
Treatment Naïve ◽  
Stool Samples ◽  
H Pylori

Helicobacter pylori infection is mainly diagnosed non-invasively, with susceptibility testing traditionally requiring endoscopy. Treatment is empiric, with clarithromycin triple therapy recommended where resistance rates are below 15%. Rising clarithromycin resistance resulting in high therapy failure rates is seen worldwide but United States data is limited. We developed a real-time PCR assay for simultaneous detection of H. pylori and genotypic markers of clarithromycin resistance directly from stool specimens. The assay was validated by testing 524 stool samples using an H. pylori stool antigen test as the reference method for detection accuracy and Sanger sequencing to confirm genotypic susceptibility results. A separate set of 223 antigen positive stool samples was tested and retrospective medical record review conducted to define clinical utility. PCR resulted in 88.6% and 92.8% sensitivity in the validation and clinical study sets, respectively. Sequencing confirmed correct detection of clarithromycin resistance-associated mutations in all positive validation samples. The PCR predicted clarithromycin resistance rate was 39% in the clinical data set overall and 28% in treatment naïve patients; the clarithromycin triple therapy eradication rate in treatment naïve patients was 62%. The clarithromycin triple therapy success was lower when resistance was predicted by PCR (41%) than when no resistance was predicted (70%, p=0.03). PCR was positive in 98% of antigen positive stools from patients tested for eradication. The described PCR assay can accurately and non-invasively diagnose H. pylori, provide genotypic susceptibility, and test for eradication. Our findings support the need for susceptibility-guided therapy in our region if a clarithromycin-based regimen is considered.

scholarly journals Evaluation of DNA extraction methods for quantifying Helicobacter pylori in water with PCR inhibitors

2021 ◽  
Author(s):  
Eun-Sook Lee ◽  
So-Yang Cha ◽  
Jong-Soon Jung
Keyword(s):  
Helicobacter Pylori ◽  
Real Time ◽  
Dna Extraction ◽  
Water Samples ◽  
Real Time Pcr ◽  
Heavy Rain ◽  
Extraction Methods ◽  
Pcr Inhibitors ◽  
H Pylori ◽  
Dna Extraction Methods

Abstract DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water.

scholarly journals Helicobacter pylori Eradication According to Sequencing-Based 23S Ribosomal RNA Point Mutation Associated with Clarithromycin Resistance

2019 ◽  
Vol 9 (1) ◽  
pp. 54 ◽  
Author(s):  
Seung In Seo ◽  
Byoung Joo Do ◽  
Jin Gu Kang ◽  
Hyoung Su Kim ◽  
Myoung Kuk Jang ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Point Mutation ◽  
Ribosomal Rna ◽  
Eradication Rate ◽  
Point Mutations ◽  
Clarithromycin Resistance ◽  
Clinically Significant ◽  
H Pylori ◽  
23S Ribosomal Rna ◽  
Significant Point

Background/Aims: Clarithromycin resistance in Helicobacter pylori is associated with point mutations in the 23S ribosomal RNA (rRNA) gene. We investigated the point mutations in the 23S rRNA genes of patients with clarithromycin-resistant H. pylori and compared the H. pylori eradication rates based on the point mutations. Methods: A total of 431 adult patients with H. pylori infection were recruited in Kangdong Sacred Heart Hospital in 2017 and 2018. Patients who did not have point mutations related to clarithromycin resistance and/or had clinically insignificant point mutations were treated with PAC (proton pump inhibitor, amoxicillin, clarithromycin) for seven days, while patients with clinically significant point mutations were treated with PAM (proton pump inhibitor, amoxicillin, metronidazole) for seven days. H. pylori eradication rates were compared. Results: Sequencing-based detection of point mutations identified four mutations that were considered clinically significant (A2142G, A2142C, A2143G, A2143C). The clarithromycin resistance rate was 21.3% in the overall group of patients. A2143G was the most clinically significant point mutation (84/431, 19.5%), while T2182C was the most clinically insignificant point mutation (283/431, 65.7%). The overall H. pylori eradication rate was 83.7%, and the seven-day PAM-treated clarithromycin-resistance group showed a significantly lower eradication rate than the seven-day PAC-treated nonresistance group (ITT; 55.4% (51/92) vs. 74.3% (252/339), p = 0.001, PP; 66.2% (51/77) vs. 88.4% (252/285), p = 0.0001). Conclusions: There were significantly lower eradication rates in the patients with clarithromycin-resistant H. pylori when treated with PAM for seven days. A future study comparing treatment regimens in clarithromycin-resistant H. pylori-infected patients may be necessary.

scholarly journals Phenotypic and Genotypic Analysis of Resistant Helicobacter pylori Strains Isolated from Children with Gastrointestinal Diseases

Diagnostics ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 759
Author(s):  
Monika Maria Biernat ◽  
Aldona Bińkowska ◽  
Łukasz Łaczmański ◽  
Paweł Biernat ◽  
Paweł Krzyżek ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antibiotic Resistance ◽  
Point Mutations ◽  
Gastrointestinal Diseases ◽  
Drug Susceptibility Testing ◽  
Test Method ◽  
Ulcer Disease ◽  
Genotypic Analysis ◽  
Clinical Diagnoses ◽  
H Pylori

Antibiotic resistance of Helicobacter pylori is currently a global issue. The aim of this study was to analyze actual antibiotic resistance rates of H. pylori strains isolated from children with primary infections and to compare the incidence of mutations that determine resistance to clarithromycin (CH) and metronidazole (MET) in children with different clinical diagnoses. A total of 91 H. pylori strains were isolated from 108 children with primary infections. Drug susceptibility testing of the strains was performed using E-test method. Classical sequencing of DNA fragments was used to detect point mutations for CH and MET resistance. Resistance to CH was detected in 31% of isolated strains (28/91), while resistance to MET and CH was detected in 35% (32/91) of strains. A2143G was the most frequently detected mutation and was dominant among strains isolated from children with peptic ulcer disease (80%). Mutations in the rdxA gene were found significantly more frequently among MET-resistant strains than MET-sensitive strains (p = 0.03, Chi2 = 4.3909). In children, a higher frequency of H. pylori multiresistant strains was observed compared with the previous study in the same area. Differences were found in the occurrence of point mutations among H. pylori strains resistant to CH isolated from children with different clinical diagnoses.

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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