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Maghami AA, Mobarez AM, Yadegar A, Nikkhah M, Sadeghi A, Mousazadeh M. Evaluation of optimized Real time-PCR HRM assay and SPR-based biosensor for noninvasive isolation of H. pylori and Clarithromycin resistance 23S-SNP subtype. Diagn Microbiol Infect Dis 2025; 111:116722. [PMID: 39919347 DOI: 10.1016/j.diagmicrobio.2025.116722] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2024] [Revised: 01/23/2025] [Accepted: 01/29/2025] [Indexed: 02/09/2025]
Abstract
High Resolution Melting analysis is a highly sensitive molecular method, and the plasmonic-based sensor is a convenient test, be clinically elicited, in simultaneous H. pylori control and the prevention of Clarithromycin resistant strains related to SNPs. Comparing gold standard tests, we evaluated HRMA and the SPR-based biosensor in regard to noninvasive forms of H. pylori management, and explored resistance through a possible mechanism by 23S-SNPs.We optimized Realtime-PCR HRM analysis, and SPR-based biosensor. Referring to CTs ± 38, isolation sensitivity was evaluated at 74 %. Compared to the reference-test, results agreements were assessed with a kappa of 43 %, p = 0.001. Within melting plots (differentiation plot), the strains were detectable at Tms ranging from 81.5 to 82.3 (°C). Contributing to phenotypically determined resistant strains for 44 %, there was a wide distribution among resistant variants assessed through Tms of 82, 81.5, 82, 82.3, and 81.7 (°C). A2143C was the only mutation isolated at a specific Tm of 82.3°C; kappa = 8 %, p = 0.3. For sensing analysis, including 100 % specificity, this sensor type was associated with lower sensitivity of 49 %, kappa=19 %, p = 0. 01. For SNP, specificity, kappa value, and p-value of isolation were 100 %, 100 %, and 0.001. Lod was 0.003 ng/mL and 0.03 µg/mL for HRM assay and sensing analysis. In our evaluations, applicable priority of HRM analysis was the higher estimation of sensitivity; its differentiation plot was completely covered the results of strain variability regarding specific SNPs detection. Consistent with the biosensor for isolation of SNP, the development of biosensors is the necessity for noninvasive H. pylori detection.
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Affiliation(s)
- Atena Abedi Maghami
- Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Ashraf Mohabati Mobarez
- Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
| | - Abbas Yadegar
- Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Maryam Nikkhah
- Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran; Department of Sensor and Biosensor, Faculty of Interdisciplinary Sciences and Technologies, Tarbiat Modares University, Tehran, Iran
| | - Amir Sadeghi
- Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical, Sciences, Tehran, Iran
| | - Marziyeh Mousazadeh
- Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
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Chakraborty S, Rana S, Gulati S, Chaudhary S, Panigrahi MK, Hallur VK, Maiti S, Chakraborty D, Makharia GK. Engineered FnCas9 mediated mutation profiling for clarithromycin resistance in Helicobacter pylori strains isolated from Indian patients with gastrointestinal disorders. Microchem J 2024; 207:112051. [DOI: 10.1016/j.microc.2024.112051] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2025]
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Keikha M, Karbalaei M. Prevalence of antibiotic heteroresistance associated with Helicobacter pylori infection: A systematic review and meta-analysis. Microb Pathog 2022; 170:105720. [PMID: 35964816 DOI: 10.1016/j.micpath.2022.105720] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2022] [Revised: 08/05/2022] [Accepted: 08/08/2022] [Indexed: 10/15/2022]
Abstract
BACKGROUND Heteroresistance is a general term to describe diverse responses to specific antibiotics that can occur due to infection with either multiple bacterial strains or microevolution of a single strain during chronic infection. Due to limited information regarding heteroresistance Helicobacter pylori (H. pylori) infection, the current study was carried out to evaluate the prevalence of this phenomenon. METHODS For this study, all potential relevant studies were collected by searching international databases such as ISI Web of Science, PubMed, Scopus, ScienceDirect, ProQuest, Embase, DOAJ, China National Knowledge Infrastructure (CNKI), and Google Scholar. Finally, the frequency of heteroresistance H. pylori infection was measured using the event rate corresponding to 95% confidence intervals. RESULTS A total of 26 studies met our criteria; the eligible studies were related to the years 2001-2022. Our results showed that the prevalence of heteroresistance H. pylori strains was 60.1% to clarithromycin, 61.1% to metronidazole, 46.1% to levofloxacin, 3.8% to amoxicillin, and 21.1% to tetracycline. Our literature review also showed discrepancy of antimicrobial susceptibility test in strains isolated from different anatomical sites of the stomach. Heteroresistance H. pylori infection in developing countries is mostly due to infection with multiple H. pylori strains, while in developed countries it is due to microevolution of a single H. pylori strain in response to antibiotic pressure. CONCLUSIONS Heteroresistance H. pylori infection interferes with successful therapy and eventually can lead to the treatment failure. If a biopsy is taken from only one gastric site, resistant strains of H. pylori may be underestimated. Considering the role of heteroresistance H. pylori infection in treatment failure, it is very important for gastroenterologists to improve their knowledge about this fact. Regardingly, new guidelines should be developed and designed for the management and treatment of heteroresistance H. pylori infection.
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Affiliation(s)
- Masoud Keikha
- Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Mohsen Karbalaei
- Department of Microbiology and Virology, School of Medicine, Jiroft University of Medical Sciences, Jiroft, Iran.
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Wang YH, Gong XL, Liu DW, Zeng R, Zhou LF, Sun XY, Liu DS, Xie Y. Characteristics of Helicobacter pylori Heteroresistance in Gastric Biopsies and Its Clinical Relevance. Front Cell Infect Microbiol 2022; 11:819506. [PMID: 35186783 PMCID: PMC8855363 DOI: 10.3389/fcimb.2021.819506] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2021] [Accepted: 12/20/2021] [Indexed: 11/13/2022] Open
Abstract
BackgroundAntimicrobial susceptibility testing (AST) plays a vital role in anti-Helicobacter pylori treatment, but the traditional AST method has difficulty detecting heteroresistance, which may cause an increased prevalence of resistant strains and eradication failure.AimsTo investigate the characteristics of heteroresistance in H. pylori in gastric biopsies and investigate its clinical relevance.MethodA total of 704 gastric biopsies were selected for 23S rRNA and gyrA gene sequencing, 470 H. pylori isolates from these biopsies were selected for AST, and the clinical characteristics of the patients were reviewed.ResultFor the 699 biopsies that were positive for 23S rRNA gene, 98 (14.0%) showed a heteroresistance genotype, and a wild type (WT) combined with A2143G (86.7%) genotype was found in most samples. For the 694 biopsies that were positive for gyrA gene, 99 (14.3%) showed a heteroresistance genotype, and a WT combined with 87K (26.3%) or WT combined with 91N (23.2%) genotype was predominant. According to the E-test results, the resistance rates of heteroresistance genotype samples for clarithromycin and levofloxacin were 36.2% and 68.1%, respectively. When dividing the heteroresistance samples into different groups according to the sequencing profile peaks of the mutation position, the resistance rates were higher along with mutation peaks at the mutation position. In addition, patients infected with mutated or heteroresistant strains showed lower peptic ulcer detection rates than those infected with the WT strain (p < 0.05).ConclusionHeteroresistance genotypes for clarithromycin and levofloxacin were not rare in H. pylori. Most cases with a heteroresistance genotype showed a susceptible phenotype for clarithromycin and a resistance phenotype for levofloxacin. Patients infected with heteroresistance genotype strains showed a lower peptic ulcer detection rate than those infected with the WT strain.
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Affiliation(s)
- You-hua Wang
- Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, China
| | - Xiao-ling Gong
- Department of Blood Transfusion, The First Affiliated Hospital of Nanchang University, Nanchang, China
| | - Ding-wei Liu
- Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, China
| | - Rong Zeng
- Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, China
| | - Lin-fu Zhou
- Department of Biochemistry, Department of the Children’s Hospital, National Clinical Research Center for Child Health, Zhejiang University School of Medicine, Hangzhou, China
| | - Xiao-yan Sun
- Department of Epidemiology, Bethune International Peace Hospital, Shijiazhuang, China
| | - Dong-sheng Liu
- Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, China
- *Correspondence: Dong-sheng Liu, ; Yong Xie,
| | - Yong Xie
- Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, China
- *Correspondence: Dong-sheng Liu, ; Yong Xie,
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Gong RJ, Xu CX, Li H, Liu XM. Polymerase chain reaction-based tests for detecting Helicobacter pylori clarithromycin resistance in stool samples: A meta-analysis. World J Clin Cases 2021; 9:133-147. [PMID: 33511178 PMCID: PMC7809662 DOI: 10.12998/wjcc.v9.i1.133] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/06/2020] [Revised: 11/07/2020] [Accepted: 11/14/2020] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Helicobacter pylori (H. pylori) infection is closely associated with the etiology of a variety of gastric diseases. The effective eradication of H. pylori infection has been shown to reduce the incidence of gastric carcinoma. However, the rate of H. pylori eradication has significantly declined due to its increasing resistance to antibiotics, especially to clarithromycin. Therefore, the detection of clarithromycin resistance is necessary prior to the treatment of H. pylori. Although many studies have been conducted on the use of polymerase chain reaction (PCR)-based tests to detect clarithromycin resistance in stool samples, no accurate data on the feasibility of these tests are available. Here, we performed a meta-analysis to assess the feasibility of these noninvasive tests.
AIM To evaluate the reliability of PCR-based tests for detecting H. pylori clarithromycin resistance in stool samples.
METHODS We searched PubMed, Medline, Embase, and other databases for articles that evaluated the value of the PCR analysis of stool samples for detecting the resistance of H. pylori to clarithromycin. We collected cross-sectional studies that met the inclusion criteria. Diagnostic accuracy measures were pooled using a random-effects model. The risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. Subgroup analysis was also conducted according to PCR type, purification technique, reference standard, mutation site, sample weight, number of patients, and age group, and the clinical utility of diagnostic tests was evaluated using the Likelihood Ratio Scatter Graph.
RESULTS Out of the 1818 identified studies, only 11 met the eligibility criteria, with a total of 592 patients assessed. A meta-analysis of the random-effect model showed that PCR-based analysis of stool samples had high diagnostic accuracy for detecting clarithromycin resistance in patients infected with H. pylori. The combined sensitivity was 0.91 [95% confidence interval (CI): 0.83-0.95], Q = 30.34, and I2 = 67.04, and the combined specificity was 0.97 (95%CI: 0.62-1.00), Q = 279.54, and I2 = 96.42. The likelihood ratio for a positive test was 33.25 (95%CI: 1.69-652.77), and that for a negative test was 0.10 (95%CI: 0.05-0.18), with an area under the curve of 0.94. The diagnostic odds ratio was 347.68 (95%CI: 17.29-6991.26). There was significant statistical heterogeneity, and the sub-analyses showed significant differences in the number of patients, sample weight, purification methods, PCR types, mutation points, and reference standards. The included studies showed no risk of publication bias.
CONCLUSION PCR-based tests on stool samples have high diagnostic accuracy for detecting H. pylori clarithromycin resistance.
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Affiliation(s)
- Ren-Jie Gong
- Department of Gastroenterology, The Third Xiangya Hospital of Central South University, Changsha 410013, Hunan Province, China
| | - Can-Xia Xu
- Department of Gastroenterology, The Third Xiangya Hospital of Central South University, Changsha 410013, Hunan Province, China
| | - Huan Li
- Department of Gastroenterology, The Third Xiangya Hospital of Central South University, Changsha 410013, Hunan Province, China
| | - Xiao-Ming Liu
- Department of Gastroenterology, The Third Xiangya Hospital of Central South University, Changsha 410013, Hunan Province, China
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Marques AT, Vítor JMB, Santos A, Oleastro M, Vale FF. Trends in Helicobacter pylori resistance to clarithromycin: from phenotypic to genomic approaches. Microb Genom 2020; 6:e000344. [PMID: 32118532 PMCID: PMC7200067 DOI: 10.1099/mgen.0.000344] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2019] [Accepted: 02/10/2020] [Indexed: 12/15/2022] Open
Abstract
For a long time Helicobacter pylori infections have been treated using the macrolide antibiotic, clarithromycin. Clarithromycin resistance is increasing worldwide and is the most common cause of H. pylori treatment failure. Here we review the mechanisms of antibiotic resistance to clarithromycin, detailing the individual and combinations of point mutations found in the 23S rRNA gene associated with resistance. Additionally, we consider the methods used to detect clarithromycin resistance, emphasizing the use of high-throughput next-generation sequencing methods, which were applied to 17 newly sequenced pairs of H. pylori strains isolated from the antrum and corpus of a recent colonized paediatric population. This set of isolates was composed of six pairs of resistant strains whose phenotype was associated with two point mutations found in the 23S rRNA gene: A2142C and A2143G. Other point mutations were found simultaneously in the same gene, but, according to our results, it is unlikely that they contribute to resistance. Further, among susceptible isolates, genomic variations compatible with mutations previously associated with clarithromycin resistance were detected. Exposure to clarithromycin may select low-frequency variants, resulting in a progressive increase in the resistance rate due to selection pressure.
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Affiliation(s)
- Andreia T. Marques
- Host–Pathogen Interactions Unit, Research Institute for Medicines (iMed-ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal
| | - Jorge M. B. Vítor
- Host–Pathogen Interactions Unit, Research Institute for Medicines (iMed-ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal
- Department of Biochemistry and Human Biology, Faculty of Pharmacy, Universidade de Lisboa, 1649 003 Lisbon, Portugal
| | - Andrea Santos
- National Reference Laboratory for Gastrointestinal Infections, Department of Infectious Diseases, National Institute of Health Dr Ricardo Jorge, Lisbon, Portugal
| | - Mónica Oleastro
- National Reference Laboratory for Gastrointestinal Infections, Department of Infectious Diseases, National Institute of Health Dr Ricardo Jorge, Lisbon, Portugal
| | - Filipa F. Vale
- Host–Pathogen Interactions Unit, Research Institute for Medicines (iMed-ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal
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Genotyping and antimicrobial resistance patterns of Helicobacter pylori in human and dogs associated with A2142G and A2143G point mutations in clarithromycin resistance. Microb Pathog 2018; 123:330-338. [PMID: 30031039 DOI: 10.1016/j.micpath.2018.07.016] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2018] [Revised: 07/12/2018] [Accepted: 07/13/2018] [Indexed: 02/07/2023]
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Wang YH, Li Z, Wang L, Zhu-Ge LY, Zhao RL, Wu S, Wang Y, An Y, Xie Y. A systematic review and meta-analysis of genotypic methods for detecting antibiotic resistance in Helicobacter pylori. Helicobacter 2018; 23:e12467. [PMID: 29405526 DOI: 10.1111/hel.12467] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND Antibiotic susceptibility testing is essential for tailored treatments to cure Helicobacter pylori (H. pylori) infection. However, phenotypic methods have some limitations. OBJECTIVES To evaluate the feasibility of genotypic detection methods compared with phenotypic detection methods using samples taken from H. pylori-infected patients. METHODS Literature searches were conducted in the following databases (from January 2000 to November 2016): PubMed, Embase, the Cochrane Library, and Web of Science. A meta-analysis and systematic review was performed for studies that compared genotypic methods with phenotypic methods for the detection of H. pylori antibiotic susceptibility. RESULTS This meta-analysis showed that the pooled sensitivity, specificity, and diagnostic odds ratio (DOR) for the A2142G/C and/or A2143G combination for the detection of clarithromycin resistance in the strain samples were 0.97 (95% CI: 0.94-0.99), 1.00 (95% CI: 0.99-1.00), and 13 742 (95% CI: 1708-110 554), respectively. The pooled sensitivity, specificity, and DOR for the A2142G/C and/or A2143G combination for the detection of clarithromycin resistance in biopsy samples were 0.96 (95% CI: 0.90-0.99), 0.96 (95% CI: 0.91-0.99), and 722 (95% CI: 117-4443), respectively. The summarized sensitivity, specificity, and DOR value for the ability of the genotypic methods to detect quinolone resistance in biopsy specimens were 0.97 (95% CI: 0.87-0.99), 0.99 (95% CI: 0.92-1.00), and 6042 (95% CI: 486-75 143), respectively. CONCLUSION The genotypic detection methods were reliable for the diagnosis of clarithromycin and quinolone resistance in the strain and biopsy specimens. The A2142G/C and/or A2143G combination had the best sensitivity and specificity for the detection of clarithromycin resistance.
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Affiliation(s)
- You-Hua Wang
- Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China
| | - Zhen Li
- Department of Medical College, Nanchang University, Nanchang, Jiangxi Province, China
| | - Le Wang
- Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China.,Jiangxi Provincial Key Laboratory of Translational Medicine and Oncology, Jiangxi Cancer Hospital, Nanchang, Jiangxi Province, China
| | - Li-Ya Zhu-Ge
- Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China
| | - Ru-Lin Zhao
- Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China.,Department of Medical College, Nanchang University, Nanchang, Jiangxi Province, China
| | - Shuang Wu
- Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China
| | - Ya Wang
- Department of Medical College, Nanchang University, Nanchang, Jiangxi Province, China
| | - Ying An
- Department of Medical College, Nanchang University, Nanchang, Jiangxi Province, China
| | - Yong Xie
- Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China
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Binyamin D, Pastukh N, On A, Paritsky M, Peretz A. Phenotypic and genotypic correlation as expressed in Helicobacter pylori resistance to clarithromycin and fluoroquinolones. Gut Pathog 2017. [DOI: 10.1186/s13099-017-0198-5] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
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Aksit Bıcak D, Akyuz S, Kıratlı B, Usta M, Urganci N, Alev B, Yarat A, Sahin F. The investigation of Helicobacter pylori in the dental biofilm and saliva samples of children with dyspeptic complaints. BMC Oral Health 2017; 17:67. [PMID: 28327128 PMCID: PMC5361728 DOI: 10.1186/s12903-017-0361-x] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2016] [Accepted: 03/10/2017] [Indexed: 12/25/2022] Open
Abstract
BACKGROUND The oral cavity can be an extra-gastric reservoir for Helicobacter pylori (H.pylori). This can play a role in the pathogenesis of halitosis, glossitis, recurrent aphthous stomatitis, and dental caries. The present study was conducted to detect the presence of H.pylori within the dental biofilm and in saliva samples collected from children suffering from dyspepsia and children without any gastrointestinal complaints. Associations with gastric infection, halitosis, and some oral parameters were also evaluated. METHODS Seventy children (aged between 5-16) with dyspepsia were selected for the study group and control group composed of 30 healthy children without dyspepsia were also included in the study. After detailed oral and clinical examinations for oral parameters, saliva, and supragingival dental biofilm samples were collected for 16S rRNA and 23S rRNA genes detection by real-time polymerase chain reaction (RT-PCR). The presence of gastric H.pylori was evaluated in endoscopic biopsy specimens histopathologically. Halitosis was evaluated by benzoyl-DL-arginine-naphthylamid (BANA) test. Salivary S.mutans and Lactobacilli sp. counts were also carried out by commercial kits. RESULTS H.pylori was histopathologically detected amongst 83% of the children with the dyspeptic condition. The detection rate of this bacteria in dental biofilm and saliva samples and halitosis were found relatively higher in the dyspeptic children rather than the control group (p < 0.01). Halitosis was not significantly different between dyspeptic children and those detected with H.pylori (p > 0.05). In the gastric H.pylori positive group with dyspepsia, DMFT/S and dmft/s numbers and plaque indices were found higher than the control group (p < 0.01). Only plaque indices of gastric H.pylori negative group with dyspepsia were found higher than the control group (p < 0.01). S.mutans and Lactobacilli sp. counts were not significantly different between gastric H.pylori positive and negative groups (p > 0.05). Comparing to those with negative for both genes, in children whose dental biofilm and saliva samples were positive for both 16S rRNA and 23S rRNA genes, significantly higher results for halitosis, and DMFS numbers and significantly lower results for dmfs numbers and pH values were found (p < 0.01). CONCLUSIONS Helicobacter pylori can occur in the oral cavity aside and independently from the stomach. However, the high number of bacteria in the oral cavities of children with gastric H.pylori, an association between the presence of H.pylori and halitosis, DMFS, and pH were found.
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Affiliation(s)
- Damla Aksit Bıcak
- Faculty of Dentistry, Department of Pediatric Dentistry, Marmara University, Basibuyuk Yolu 9/3 34854 Basibuyuk, Maltepe, Istanbul, Turkey
| | - Serap Akyuz
- Faculty of Dentistry, Department of Pediatric Dentistry, Marmara University, Basibuyuk Yolu 9/3 34854 Basibuyuk, Maltepe, Istanbul, Turkey
| | - Binnur Kıratlı
- Faculty of Engineering, Department of Genetics and Bioengineering, Yeditepe University, İstanbul, Turkey
| | - Merve Usta
- Department of Pediatric Gastroenterology, Sisli Hamidiye Etfal Education and Research Hospital, İstanbul, Turkey
| | - Nafiye Urganci
- Department of Pediatric Gastroenterology, Sisli Hamidiye Etfal Education and Research Hospital, İstanbul, Turkey
| | - Burcin Alev
- Faculty of Dentistry, Department of Basic Medical Sciences, Biochemistry, Marmara University, İstanbul, Turkey
| | - Aysen Yarat
- Faculty of Dentistry, Department of Basic Medical Sciences, Biochemistry, Marmara University, İstanbul, Turkey
| | - Fikrettin Sahin
- Faculty of Engineering, Department of Genetics and Bioengineering, Yeditepe University, İstanbul, Turkey
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Navarro-Jarabo JM, Fernández-Sánchez F, Fernández-Moreno N, Hervas-Molina AJ, Casado-Caballero F, Puente-Gutierrez JJ, Pallares-Manrique H, Rodríguez-Ramos C, Fernández-Gutierrez C, Pérez-Aisa A, Rivas-Ruiz F, Montiel Quezel-Guerraz N. Prevalence of Primary Resistance of Helicobacter pylori to Clarithromycin and Levofloxacin in Southern Spain. Digestion 2017; 92:78-82. [PMID: 26227669 DOI: 10.1159/000435949] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/17/2014] [Accepted: 06/14/2015] [Indexed: 02/04/2023]
Abstract
BACKGROUND The eradication of Helicobacter pylori (HP) using clarithromycin (CLA)-based triple therapy depends on the resistance of HP to antibiotics. The Maastricht III conference recommends the implementation of locoregional surveillance programmes for primary resistance of HP to CLA. In Andalusia, there are no previous data in this respect. The aim of this study was to determine the prevalence of the primary resistance of HP to CLA and levofloxacin (LF) in southern Spain. METHODS Multicentre cross sectional study was carried out in 6 hospitals in Andalusia. Patients of both sexes numbering 401 were included (male 48%), aged 18-80 years and naïve to HP eradication. Resistance of HP to CLA (CLAr) and LF (LFr) was assessed by determining mutations by PCR: mutations of the 23S rRNA gene define CLAr and mutations of the gene gyrA define LFr. Four hundred one gastric samples were collected. CLAr was detected in 72 patients (17.9%) and LFr was detected in 56 patients (13.9%). Heteroresistance was detected for both antibiotics: CLA 37/72 (51.3%) and LF 28/56 (50%). Variability for CLAr was detected among the centres, ranging from 11.5% to 24.7% without statistical significance (p = 0.12). Female sex was related to CLAr. CONCLUSIONS In Andalusia, there is a high rate of primary CLAr and LFr. CLA-based triple therapy should be avoided as the primary eradication regimen in this region. There is a wide variability in the rate of CLAr among centres.
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Mégraud F, Bénéjat L, Ontsira Ngoyi EN, Lehours P. Molecular Approaches to Identify Helicobacter pylori Antimicrobial Resistance. Gastroenterol Clin North Am 2015; 44:577-96. [PMID: 26314669 DOI: 10.1016/j.gtc.2015.05.002] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/21/2023]
Abstract
Antimicrobial susceptibility testing is needed to adapt Helicobacter pylori treatment to obtain the best results. Beside the standard phenotypic methods, molecular methods are increasingly used. The value of these molecular tests is that they are quick, independent of the transport conditions, easy to standardize, and commercial kits are available. In this article, these methods are reviewed, focusing on the determination of H pylori resistance to macrolides and fluoroquinolones, and mentioning also the methods used for tetracycline and rifampin.
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Affiliation(s)
- Francis Mégraud
- Bacteriology Laboratory, INSERM U853, University of Bordeaux, Bordeaux F-33000, France.
| | - Lucie Bénéjat
- Bacteriology Laboratory, INSERM U853, University of Bordeaux, Bordeaux F-33000, France
| | | | - Philippe Lehours
- Bacteriology Laboratory, INSERM U853, University of Bordeaux, Bordeaux F-33000, France
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Improved allele-specific PCR assays for detection of clarithromycin and fluoroquinolone resistant of Helicobacter pylori in gastric biopsies: identification of N87I mutation in GyrA. Diagn Microbiol Infect Dis 2014; 81:251-5. [PMID: 25600075 DOI: 10.1016/j.diagmicrobio.2014.12.003] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2014] [Revised: 12/01/2014] [Accepted: 12/10/2014] [Indexed: 12/18/2022]
Abstract
Molecular testing can rapidly detect Helicobacter pylori susceptibility using gastric biopsies. Allele-specific polymerase chain reaction (ASP-PCR) was used to identify H. pylori 23S rRNA and gyrA mutation using gastric biopsies from Colombian patients and confirmed by PCR and sequencing of the 23S rRNA and gyrA genes. The sensitivity and specificity of ASP-PCR were compared with susceptibilities measured by agar dilution. Samples included gastric biopsies from 107 biopsies with H. pylori infections and 20 H. pylori negative. The sensitivity and specificity of ASP-PCR for the 23S rRNA gene were both 100%. The sensitivity and specificity of ASP-PCR for the gyrA gene, published in 2007 by Nishizawa et al., were 52% and 92.7%, respectively; the lower sensitivity was due to the presence of mutation N87I in our samples, which were not detected by the test. In this study, we designed new primers to detect the mutation N87I in GyrA. The ASP-PCR was performed with the original primers plus the new primers. The molecular test with the new primers improved the sensitivity to 100%. In conclusion, ASP-PCR provides a specific and rapid means of predicting resistance to clarithromycin and levofloxacin in gastric biopsies.
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Jahani Moghadam F, Navidifar T, Amin M. Antibacterial Activity of Garlic (Allium sativum L.) on Multi-Drug Resistant Helicobacter pylori Isolated From Gastric Biopsies. INTERNATIONAL JOURNAL OF ENTERIC PATHOGENS 2014. [DOI: 10.17795/ijep16749] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
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Trespalacios AA, Otero W, Caminos JE, Mercado MM, Ávila J, Rosero LE, Arévalo A, Poutou-Piñales RA, Graham DY. Phenotypic and genotypic analysis of clarithromycin-resistant Helicobacter pylori from Bogotá D.C., Colombia. J Microbiol 2013; 51:448-52. [DOI: 10.1007/s12275-013-2465-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2012] [Accepted: 02/20/2013] [Indexed: 12/21/2022]
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Xiong LJ, Tong Y, Wang Z, Mao M. Detection of clarithromycin-resistant Helicobacter pylori by stool PCR in children: a comprehensive review of literature. Helicobacter 2013; 18:89-101. [PMID: 23067446 DOI: 10.1111/hel.12016] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
BACKGROUND Helicobacter pylori infection is acquired mainly during childhood. To eradicate H. pylori, clarithromycin-based triple therapy has been recommended in children and adults by the latest Maastricht Consensus. However, the prevalence of clarithromycin-resistant H. pylori was higher in children than that in adults. Therefore, rapid, reliable and noninvasive methods for detecting clarithromycin-resistant H. pylori strains should be developed for children. MATERIALS AND METHODS Studies on evaluating stool PCR in detecting clarithromycin-resistant H. pylori and epidemiological surveys of the prevalence of clarithromycin-resistant H. pylori in children were searched in PubMed (from 1966 to December, 2011) for reviewing. RESULTS The average rates of primary clarithromycin-resistant H. pylori ranged from less than 10% to more than 40% in different regions. The rates of secondary resistance to clarithromycin were higher than primary resistance in the same population. In H. pylori isolated from children, the frequent point mutations that are responsible for the clarithromycin resistance included A2143G, A2142G, A2142C and A2144G, and they varied geographically. Comparing with culture-based susceptibility tests, stool PCR performed excellently for their rapidity, independence of bacterial growth, reproducibility and easy standardization. However, stool PCR showed lower sensitivity but perfect specificity in detection of clarithromycin-resistant H. pylori in children. Methodology and mixed infections of resistant H. pylori strains might contribute to the considerable discrepancies of stool PCR results. CONCLUSION Detection of clarithromycin-resistant H. pylori by stool PCR for children are reliable, rapid, noninvasive methods that are worthy of further clinical promotion. However, more evaluations of stool PCR in detection of clarithromycin-resistant H. pylori in children need to be conducted.
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Affiliation(s)
- Li Jing Xiong
- Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, China
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Detection of four clarithromycin resistance point mutations in Helicobacter pylori: comparison of real-time PCR and PCR-RFLP methods. ACTA ACUST UNITED AC 2012. [DOI: 10.1007/s00580-012-1519-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
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Khedmat H, Karami A, Safiri Z, Amini M, Bakhtiari A, Karbasi A, Jayhounian M, Jalalian H, Taheri S. Helicobacter pylori genotypes can predict gastric tissue histopathology: a longitudinal study of Iranian patients. J Infect Public Health 2012; 5:153-158. [PMID: 22541262 DOI: 10.1016/j.jiph.2011.10.009] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2011] [Revised: 08/22/2011] [Accepted: 10/10/2011] [Indexed: 01/25/2023] Open
Abstract
INTRODUCTION Several factors have been suggested to account for differences in the virulence of Helicobacter pylori infections in various populations. Evidence suggests the existence of different strains of H. pylori with different degrees of virulence. The present study aimed to investigate the gastric histopathology in Iranian patients infected with H. pylori and to investigate the relationship between the severity of gastritis and four different bacterial virulence-associated genotypes. METHODS AND MATERIALS All of the patients with positive results from a pathological examination, a rapid urease test, and PCR analysis for H. pylori infection were consecutively included into the study. The classification and grading of gastritis were performed according to the Sydney System. Esophagitis was classified endoscopically according to the Savary-Miller grading system. The primers used in this study targeted 16S rRNa (521 bp), Urease A (411 bp), Cag A (400 bp), and 26 kDa (303 bp). RESULTS Twenty-eight patients were included in the study. The presence of Cag A showed a significant relationship with higher gastritis grades (3.0±0.7 vs. 2.3±0.9, p=0.024) and higher scores for H. pylori infection (3.0±0.7 vs. 2.3±0.7, p=0.027). The patients infected with 26 kDa-positive H. pylori had significantly higher infection scores (3.5±0.6 vs. 2.5±0.6, p=0.020). CONCLUSION This study showed that CagA-positive H. pylori infection is associated with more severe gastritis and with increased bacterial density and inflammation in the biopsy specimens. The 303-bp positive genotype was also significantly associated with higher grades of esophagitis. Additional in-depth trials will be helpful in extending our findings.
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Affiliation(s)
- Hossein Khedmat
- Baqiyatallah Research Center for Gastroenterology & Liver Diseases, Tehran, Iran.
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Kargar M, Ghorbani-Dalini S, Doosti A, Souod N. Real-time PCR for Helicobacter pylori quantification and detection of clarithromycin resistance in gastric tissue from patients with gastrointestinal disorders. Res Microbiol 2012; 163:109-13. [DOI: 10.1016/j.resmic.2011.11.005] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2011] [Accepted: 11/16/2011] [Indexed: 12/21/2022]
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Liou JM, Chen CC, Chen MJ, Chang CY, Fang YJ, Lee JY, Sheng WH, Wang HP, Wu MS, Lin JT. Empirical modified sequential therapy containing levofloxacin and high-dose esomeprazole in second-line therapy for Helicobacter pylori infection: a multicentre clinical trial. J Antimicrob Chemother 2011; 66:1847-52. [PMID: 21632579 DOI: 10.1093/jac/dkr217] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
OBJECTIVES Sequential therapy appears to achieve a higher Helicobacter pylori eradication rate than triple therapy. We assessed the efficacy and tolerability of modified sequential therapy containing levofloxacin and high-dose esomeprazole in second-line therapy. METHODS Patients who failed first-line triple therapy with clarithromycin, amoxicillin and a proton pump inhibitor were eligible in this multicentre trial. Eligible patients were treated with esomeprazole 40 mg and amoxicillin 1 g for the first 5 days, followed by esomeprazole 40 mg, levofloxacin 250 mg and metronidazole 500 mg for another 5 days (all given twice daily). Eradication was confirmed with a (13)C-urea breath test 6 weeks after therapy. Drug susceptibility, presence/absence of gyrA mutation and CYP2C19 genotype were also determined. RESULTS A total of 142 patients were enrolled. The eradication rate was 95.1% [135/142, 95% confidence interval (CI) 91.5%-98.6%] in the intention-to-treat analysis and 96.4% (133/138, 95% CI 93.3%-99.5%) in the per protocol analysis. Four patients (2.8%) failed to take at least 80% of the drugs due to adverse effects. The eradication rates were 50% (4/8) and 97.7% (43/44) in patients with and without metronidazole resistance, respectively (P = 0.001). The eradication rates were 84.6% (11/13) and 95.1% (58/61) in patients with and without gyrA mutation, respectively (P = 0.210). The eradication rates were not affected by the CYP2C19 polymorphism (P = 0.421). CONCLUSIONS This modified sequential therapy achieved an excellent eradication rate (>95%) in second-line treatment and the eradication rate appeared to be affected by metronidazole resistance.
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Affiliation(s)
- Jyh-Ming Liou
- Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan
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Abstract
Despite the fact that sequential therapy has been evaluated in more than 2500 patients and has been shown to on average provide Helicobacter pylori eradication in 90% to 94%, some authorities still question whether it should be a first-line anti-H. pylori regimen. Here, we discuss H. pylori eradication using experience and expectations with other common bacterial infections as a frame of reference. H. pylori is no exception and near 100% success is expected for optimized regimens treating susceptible infections. As such, the proper comparator would be the relation to 100% eradication. Superiority to another, often proven inferior, therapy per se provides little or no useful information. Treatment failures in infectious diseases are typically easily explainable and most often relate to the presence of antimicrobial resistance or failure to take the drugs. We provide a model for predicting the results of H. pylori combination therapies in relation to the pattern and prevalence of resistance. The results are consistent with clinical practice and explain why sequential is typically superior and essentially never inferior to triple therapy. We also show when meta-analysis is an inappropriate technique for the analysis of H. pylori clinical trials and discuss how to appropriately use the technique. Finally, we discuss why the location of studies (eg, Italy), is unimportant and explain why, from the standpoint of a therapy for an infectious disease, sequential therapy is a significant advance and should be considered one of the replacements for the outdated legacy triple therapy (proton pump inhibitor--clarithromycin--amoxicillin).
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Affiliation(s)
- David Y Graham
- Department of Medicine, Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Houston, Texas 77030, USA.
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Yakoob J, Jafri W, Abbas Z, Abid S, Naz S, Khan R, Khalid A. Risk factors associated with Helicobacter pylori infection treatment failure in a high prevalence area. Epidemiol Infect 2011; 139:581-590. [PMID: 20525411 DOI: 10.1017/s0950268810001226] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Triple therapy is commonly used for the treatment of Helicobacter pylori infection. We determined risk factors associated with its failure in compliant patients focusing on H. pylori density, virulence marker and 23S ribosomal RNA (rRNA) point mutations associated with clarithromycin resistance. H. pylori infection was diagnosed by 14C urea breath test (14C UBT) and rapid urease test or histology. Triple therapy with esomeprazole 20 mg b.i.d., amoxicillin 1 g b.i.d. and clarithromycin 500 mg b.i.d. was prescribed for 10 days. 14C UBT was repeated 4 weeks after treatment. In total, 111 patients [69 (62%) males] with a mean age of 46±16 years were enrolled. The mean age of treatment failure was 39±14 years compared to 48±16 years with eradication (P=0·002). Treatment failure was associated with younger mean age, point mutations in the 23S rRNA gene of H. pylori and vacA s1a and m1 when associated with cagA negativity.
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Affiliation(s)
- J Yakoob
- Department of Medicine, The Aga Khan University, Karachi, Pakistan.
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Abstract
Although Helicobacter pylori infection is both a common and a serious bacterial infection, antimicrobial therapies have rarely been optimized, are prescribed empirically, and provide inferior results compared with antimicrobial therapies for other common infectious diseases. The effectiveness of many of the frequently recommended H. pylori infection treatment regimens has been increasingly compromised by antimicrobial resistance. Regional data on the susceptibility of strains of H. pylori to available antimicrobials are sorely needed. Noninvasive molecular methods are possible to assess clarithromycin susceptibility in isolates obtained from stool specimens. As a general rule, clinicians should prescribe therapeutic regimens that have a ≥90% or, preferably, ≥95% eradication rate locally. If no available regimen can achieve a ≥90% eradication rate, clinicians should use the most effective regimen(s) available locally. Eradication of infection should always be confirmed after treatment in order to provide feedback regarding local effectiveness and an early warning of increasing resistance. In most regions of the world, four-drug treatment regimens, including a PPI plus three antimicrobials (clarithromycin, metronidazole/tinidazole and amoxicillin), or a PPI plus a bismuth plus tetracycline and metronidazole provide the best results. Standard triple therapy (a PPI, amoxicillin and clarithromycin) should now be avoided owing to increasing resistance to this treatment.
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Affiliation(s)
- Emiko Rimbara
- Department of Medicine, Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, 2002 Holcombe Boulevard, Houston, TX 77030, USA
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Smith SI, Fowora MA, Otegbayo JA, Abdulkareem FB, Omonigbehin EA, Adegboyega A, Contreras M, Haas R. Comparison of PCR with other diagnostic techniques for the detection of H. pylori infection in patients presenting with gastroduodenal symptons in Nigeria. INTERNATIONAL JOURNAL OF MOLECULAR EPIDEMIOLOGY AND GENETICS 2011; 2:178-184. [PMID: 21686132 PMCID: PMC3110392] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 11/10/2010] [Accepted: 01/11/2011] [Indexed: 05/30/2023]
Abstract
The study was aimed at comparing PCR methods of direct detection from biopsy using the boiling method and one other method with two known gold standards (histology and CLO test) for the diagnosis of H. pylori in Nigeria. A total of 168 biopsies (three from antrum and one from corpus each) were taken from 42 patients presenting with various gastroduodenal symptons after informed consent was obtained from them.The biopsies were analysed using the CLO test kit and histology, while the boiling method as described by Holmes and Quigley (1981) was used to obtain DNA and then PCR using the 16S rRNA gene, glmM gene and cagA gene. With CLO test 15/42 (35.71%) were positive, histology 13/42 (30.95%) were positive, 16S rRNA 22/42 (52.38%) were positive, glmM 19/42 (45.24%) were positive, cagA 19/42 (45.24%) were positive. The sensitivity and specificity of the PCR tests with CLO as the gold standard showed that the tests were 100% sensitive and varied between 74.1% to 84.1% in specificity. The PPV and NPV showed that the NPV was almost 100%, while the PPV was between 68.2% and 75%. Using the histology as the gold standard, the sensitivity was almost 100% while the specificity, the PPV were reduced in comparison to the CLO test. The PCR test using the glmM gene appears to be the most reliable test for diagnosis of H. pylori in Nigeria most especially where culture is difficult due to the power outages.
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Ben Mansour K, Burucoa C, Zribi M, Masmoudi A, Karoui S, Kallel L, Chouaib S, Matri S, Fekih M, Zarrouk S, Labbene M, Boubaker J, Cheikh I, Hriz MB, Siala N, Ayadi A, Filali A, Mami NB, Najjar T, Maherzi A, Sfar MT, Fendri C. Primary resistance to clarithromycin, metronidazole and amoxicillin of Helicobacter pylori isolated from Tunisian patients with peptic ulcers and gastritis: a prospective multicentre study. Ann Clin Microbiol Antimicrob 2010; 9:22. [PMID: 20707901 PMCID: PMC2928169 DOI: 10.1186/1476-0711-9-22] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2010] [Accepted: 08/13/2010] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND The frequency of primary resistance to antibiotics in H. pylori isolates is increasing worldwide. In Tunisia, there are limited data regarding the pattern of H. pylori antibiotic primary resistance. AIM To evaluate the primary resistance of H. pylori to clarithromycin, metronidazole and amoxicillin and to detect the mutations involved in clarithromycin resistance. MATERIALS AND METHODS 273 strains isolated from adults and children were enrolled. The primary resistance to clarithromycin, metronidazole and amoxicillin was evaluated by means of E-test minimal inhibitory concentration (MIC). The real-time PCR using Scorpion primers was performed in all cases to assess clarithromycin primary resistance and point mutations involved. RESULTS No resistance to amoxicillin was detected. For adults, resistance to clarithromycin and metronidazole was found respectively in 14.6% and 56.8%, and respectively in 18.8% and 25% in children. Overall, the rates of global primary resistance to clarithromycin and metronidazole in Tunisia were respectively determined in 15.4% and 51.3%.By the use of Scorpion PCR, the A2143G was the most frequent point mutation observed (88.1%), followed by the A2142G (11.9%); the A2142C was not found and 18 of 42 patients (42.8%) were infected by both the resistant and the susceptible genotype.The association of clarithromycin resistance with gender was not statistically significant, but metronidazole resistant strains were isolated more frequently in females (67.8%) than in males (32.2%) and the difference was significant. As for gastroduodenal diseases, the difference between strains isolated from patients with peptic ulceration and those with non peptic ulceration was not statistically significant. When about the distribution of resistant strains to clarithromycin and metronidazole between the three Tunisian cities (Tunis, Menzel Bourguiba and Mahdia), the difference was not statistically significant. CONCLUSION Local data regarding the primary resistance of H. pylori to clarithromycin, metronidazole and amoxicillin and the main genetic mutation involved in clarithromycin resistance in vivo (A2143G) are necessary to prove a clear need for a periodic evaluation of antibiotic consumption and new therapeutic strategies in Tunisia in order to avoid the emergence of resistant strains.
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Affiliation(s)
- Khansa Ben Mansour
- Microbiology laboratory/UR04SP08 Rabta University Hospital-Tunis, 1007 El Jabbari, Tunisia.
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Ho SL, Tan EL, Sam CK, Goh KL. Clarithromycin resistance and point mutations in the 23S rRNA gene in Helicobacter pylori isolates from Malaysia. J Dig Dis 2010; 11:101-5. [PMID: 20402836 DOI: 10.1111/j.1751-2980.2010.00423.x] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
OBJECTIVE To determine the prevalence of primary clarithromycin resistance amongst Helicobacter pylori (H. pylori) strains in Malaysian patients with gastroduodenal diseases, by using restriction fragment length polymorphism (RFLP) in domain V of 23S rRNA. METHODS Gastric biopsies were obtained from H. pylori positive patients undergoing gastroscopy. DNA extraction was followed by PCR amplification using the primers Hp23-1 and Hp23-2 flanking a region of 425bp within the bacterial 23S rRNA peptidyltranferase (Hp23S fragment). Analysis of the 23S rRNA gene mutations is based on the generation of restriction sites for two restriction enzymes: BbsI and BsaI, which correspond to the base substitutions characteristic of clarithromycin resistance from A to G at positions 2142 and 2143, respectively. RESULTS Gastric biopsy samples were obtained from 107 patients. A fragment of size 425bp corresponding to that expected from amplification of domain V of 23S rRNA was PCR-amplified from only 105 samples. The amplicon was subsequently subjected to restriction by BbsI and BsaI. Only 1 sample (0.95%) had the BbsI mutation (base substitution at A2142G) and 2 samples (1.90%) the BsaI mutation (base substitution at A2143G). Thus 3 of 105 (2.9%) samples harbored clarithromycin resistant strains. CONCLUSION In our experience, PCR-RFLP is a rapid and precise method to detect the resistance of H. pylori to clarithromycin. Using this method, a low prevalence of clarithromycin resistance was detected in our local Malaysian strains. This augurs well for the continued use of clarithromycin as a first line drug in the treatment and eradication of H. pylori infection.
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Affiliation(s)
- Sin-Loong Ho
- Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia
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De Francesco V, Zullo A, Ierardi E, Giorgio F, Perna F, Hassan C, Morini S, Panella C, Vaira D. Phenotypic and genotypic Helicobacter pylori clarithromycin resistance and therapeutic outcome: benefits and limits. J Antimicrob Chemother 2010; 65:327-332. [PMID: 20008044 DOI: 10.1093/jac/dkp445] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
INTRODUCTION Primary clarithromycin resistance is increasing worldwide, and it has been regarded as the main factor reducing the efficacy of Helicobacter pylori therapy. However, the clinical consequence of either phenotypic or genotypic resistance still remains unclear. This study aimed to evaluate: (i) the concordance between phenotypic (culture) and genotypic (real-time PCR) tests in assessing primary clarithromycin resistance; and (ii) the role of both in therapeutic outcome. METHODS A post hoc subgroup study was selected from a double-blind, placebo-controlled trial, enrolling 146 patients with dyspepsia or peptic ulcers never previously treated. Real-time PCR and Etest on bacterial culture for assessing clarithromycin resistance were performed. [(13)C]urea breath test (UBT), histology and rapid urease tests at entry and UBT after 4-8 weeks were used to assess infection and eradication. All patients received a 10 day therapy. RESULTS Prevalence of clarithromycin phenotypic resistance was significantly lower as compared with genotypic resistance (18.4% versus 37.6%, P < 0.001). A concordance between the two methods was present in 71.2% of cases. A significant difference in the eradication rate was seen between clarithromycin-susceptible and -resistant strains, when assessed with either Etest (92.4% versus 55.5%, P < 0.001) or a PCR-based method (94.5% versus 70.9%; P < 0.001). Of note, the eradication rate showed the lowest value (30.7%) when phenotypic bacterial resistance was genetically linked to the A2143G point mutation. CONCLUSIONS This study showed that: (i) there is a relevant discordance between the two methods; and (ii) phenotypic clarithromycin resistance markedly reduces H. pylori eradication when it is linked to a specific point mutation.
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Affiliation(s)
- Vincenzo De Francesco
- Section of Gastroenterology, Department of Medical Sciences, University of Foggia, Foggia, Italy
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Woo HY, Park DI, Park H, Kim MK, Kim DH, Kim IS, Kim YJ. Dual-priming oligonucleotide-based multiplex PCR for the detection of Helicobacter pylori and determination of clarithromycin resistance with gastric biopsy specimens. Helicobacter 2009; 14:22-8. [PMID: 19191892 DOI: 10.1111/j.1523-5378.2009.00654.x] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
BACKGROUND Assessment of Helicobacter pylori (H. pylori) clarithromycin resistance has rarely been performed routinely despite an increasing resistance rate. Our aim was to develop and evaluate the use of dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) to detect point mutations in the 23S rRNA gene responsible for clarithromycin resistance of H. pylori. MATERIALS AND METHODS Gastric biopsy specimens from 212 untreated patients with dyspepsia were examined by culture, histology, and DPO-based multiplex PCR. A disk diffusion test and E-test were used for performing phenotypic antibiotic susceptibility tests. RESULTS Among the biopsy specimens tested, 22.2% (47/212), 42.5% (90/212), and 41.5% (88/212) of the specimens were classified as H. pylori positive by culture, histology, and DPO-based multiplex PCR, respectively. Among 96 strains identified by either culture or DPO-based multiplex PCR, 80 strains were clarithromycin-susceptible and 16 strains (16.7%) were clarithromycin-resistant. There was 94.1% (32/34) concordance between phenotypic susceptibility tests and DPO-based multiplex PCR. In two patients with discrepant results, only DPO-based multiplex PCR detected clarithromycin-resistant strains. DPO-based multiplex PCR identified additional 49 clarithromycin-resistant or clarithromycin-susceptible H. pylori among 165 culture-negative specimens. CONCLUSIONS DPO-based multiplex PCR can be used as a practical method for the detection of H. pylori infection and the determination of clarithromycin susceptibility in addition to phenotypic antimicrobial susceptibility tests.
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Affiliation(s)
- Hee-Yeon Woo
- Department of Laboratory Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, South Korea
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Application of polymerase chain reaction-based assays for rapid identification and antibiotic resistance screening of Helicobacter pylori in gastric biopsies. Diagn Microbiol Infect Dis 2008; 61:67-71. [PMID: 18248939 DOI: 10.1016/j.diagmicrobio.2007.12.005] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2007] [Revised: 11/28/2007] [Accepted: 12/04/2007] [Indexed: 12/13/2022]
Abstract
The benefits of using a multiplex detection polymerase chain reaction (PCR) assay for Helicobacter pylori speciation and 2 real-time probe hybridization assays determining clarithromycin and tetracycline susceptibilities in gastric biopsies from 171 dyspeptic patients were investigated. Overall, 70 of 71 H. pylori culture-positive biopsies were PCR positive. For the 100 culture-negative biopsies, PCR identified a further 29 H. pylori positives (17% overall) and presence of resistance markers for clarithromycin (20/28) and tetracycline (2/28). The results demonstrated that PCR testing was valuable in providing improved detection rates and antibiotic susceptibility information when H. pylori culture was unsuccessful.
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Moder KA, Layer F, König W, König B. Rapid screening of clarithromycin resistance in Helicobacter pylori by pyrosequencing. J Med Microbiol 2007; 56:1370-1376. [PMID: 17893176 DOI: 10.1099/jmm.0.47371-0] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
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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Affiliation(s)
- Karen-Anja Moder
- Institute of Medical Microbiology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany
| | - Franziska Layer
- Institute of Medical Microbiology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany
| | - Wolfgang König
- Institute of Medical Microbiology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany
| | - Brigitte König
- Institute of Medical Microbiology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany
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Lin GY. Progress in research into the molecular mechanism of drug resistance of Helicobacter pylori. Shijie Huaren Xiaohua Zazhi 2007; 15:2698-2703. [DOI: 10.11569/wcjd.v15.i25.2698] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Helicobacter pylori is a pathogen of chronic and active gastritis. It is the main cause of chronic gastritis and peptic ulcer disease, and is directly related to diseases of the stomach and duodenum. This pathogen can also induce gastric carcinoma. With the extensive use of antibiotics, the number of drug resistant strains of H. pylori has rapidly increased. As a result, there are some difficulties in applying clinical therapy for diseases related to H. pylori. This paper aimed to first analyze the status and prevalence of antibiotic resistance, and then to review various drugs such as macrolides, imidazoles, tetracyclines, β-lactams and quinolones for systematically treating H. pylori infection. The mechanisms of various drug resistances, as well as detection and identification assays for typing drug resistance, are discussed. This paper presents accumulated clinical data and evidence for the clinical diagnosis and treatment of diseases related to H. pylori.
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Mégraud F, Lehours P. Helicobacter pylori detection and antimicrobial susceptibility testing. Clin Microbiol Rev 2007; 20:280-322. [PMID: 17428887 PMCID: PMC1865594 DOI: 10.1128/cmr.00033-06] [Citation(s) in RCA: 486] [Impact Index Per Article: 27.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The discovery of Helicobacter pylori in 1982 was the starting point of a revolution concerning the concepts and management of gastroduodenal diseases. It is now well accepted that the most common stomach disease, peptic ulcer disease, is an infectious disease, and all consensus conferences agree that the causative agent, H. pylori, must be treated with antibiotics. Furthermore, the concept emerged that this bacterium could be the trigger of various malignant diseases of the stomach, and it is now a model for chronic bacterial infections causing cancer. Most of the many different techniques involved in diagnosis of H. pylori infection are performed in clinical microbiology laboratories. The aim of this article is to review the current status of these methods and their application, highlighting the important progress which has been made in the past decade. Both invasive and noninvasive techniques will be reviewed.
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Affiliation(s)
- Francis Mégraud
- INSERM U853, and Université Victor Segalen Bordeaux 2, and Laboratoire de Bactériologie, Hôpital Pellegrin, Place Amélie Raba-Léon, 33076 Bordeaux cedex, France.
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Raymond J, Burucoa C, Pietrini O, Bergeret M, Decoster A, Wann A, Dupont C, Kalach N. Clarithromycin resistance in Helicobacter pylori strains isolated from French children: prevalence of the different mutations and coexistence of clones harboring two different mutations in the same biopsy. Helicobacter 2007; 12:157-63. [PMID: 17309753 DOI: 10.1111/j.1523-5378.2007.00486.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
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.
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Affiliation(s)
- Josette Raymond
- Service de Bactériologie, Hôpital Cochin-Saint Vincent de Paul, Université de Paris V, 27 rue du faubourg Saint Jacques, 75679 Paris cedex 14, France.
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Lottspeich C, Schwarzer A, Panthel K, Koletzko S, Rüssmann H. Evaluation of the novel Helicobacter pylori ClariRes real-time PCR assay for detection and clarithromycin susceptibility testing of H. pylori in stool specimens from symptomatic children. J Clin Microbiol 2007; 45:1718-22. [PMID: 17392440 PMCID: PMC1933074 DOI: 10.1128/jcm.00103-07] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The aim of the present study was to evaluate the Helicobacter pylori ClariRes assay (Ingenetix, Vienna, Austria) for the detection of H. pylori infection and the simultaneous clarithromycin susceptibility testing of the H. pylori isolates in stool samples from 100 symptomatic children. The results obtained by this novel biprobe real-time PCR method were directly compared with the results obtained from histological examination of gastric biopsy specimens, culturing, the [13C]urea breath test, and a monoclonal antibody-based stool antigen enzyme immunoassay (EIA). Fecal specimens from all 54 children who were shown to be noninfected by "gold standard" tests gave true-negative PCR results (specificity, 100%). Of the remaining 46 individuals with a positive H. pylori status, 29 were found to be positive by real-time PCR (sensitivity, 63%). For these 29 cases, the H. pylori ClariRes assay confirmed all results from phenotypic clarithromycin susceptibility testing by Etest. In summary, this investigation demonstrates that detection of Helicobacter DNA in stool samples by real-time PCR is a difficult task and that this method cannot replace the stool antigen EIA (sensitivity, 95.7%) for the accurate diagnosis of H. pylori infection in children.
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Affiliation(s)
- Christian Lottspeich
- Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-University, Pettenkoferstr. 9a, 80336 Munich, Germany
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Stege PW, Vega AE. Analysis of resistance to clarithromycin and iceA status in Helicobacter pylori isolates from San Luis, Argentina. Int J Antimicrob Agents 2006; 28:477-8. [PMID: 17046207 DOI: 10.1016/j.ijantimicag.2006.08.022] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2006] [Revised: 08/09/2006] [Accepted: 08/10/2006] [Indexed: 11/26/2022]
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de Francesco V, Margiotta M, Zullo A, Hassan C, Valle ND, Burattini O, Cea U, Stoppino G, Amoruso A, Stella F, Morini S, Panella C, Ierardi E. Primary clarithromycin resistance in Italy assessed on Helicobacter pylori DNA sequences by TaqMan real-time polymerase chain reaction. Aliment Pharmacol Ther 2006; 23:429-435. [PMID: 16423002 DOI: 10.1111/j.1365-2036.2006.02769.x] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
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=0.17) and between non-ulcer dyspepsia and peptic ulcer patients (28.4% vs. 20.7%, P=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.
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De Francesco V, Margiotta M, Zullo A, Hassan C, Troiani L, Burattini O, Stella F, Di Leo A, Russo F, Marangi S, Monno R, Stoppino V, Morini S, Panella C, Ierardi E. Clarithromycin-resistant genotypes and eradication of Helicobacter pylori. Ann Intern Med 2006; 144:94-100. [PMID: 16418408 DOI: 10.7326/0003-4819-144-2-200601170-00006] [Citation(s) in RCA: 147] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND Three point mutations (A2143G, A2142G, and A2142C) have been involved in Helicobacter pylori clarithromycin resistance. OBJECTIVE To compare the eradication rates among the different point mutations and the efficacy of triple therapy and a sequential regimen according to genotypic resistance. DESIGN Post hoc subgroup study from a multicenter, randomized trial. SETTING Two hospitals in central and southern Italy between January and December 2001. PATIENTS 156 patients with H. pylori infection. MEASUREMENTS Real-time polymerase chain reaction for assessing clarithromycin resistance; histology, rapid urease test, and 13C-urea breath test at entry and after 4 to 6 weeks. INTERVENTION 7-day triple therapy (20 mg of rabeprazole, 500 mg of clarithromycin, and 1 g of amoxicillin) in 75 patients or a 10-day sequential regimen (20 mg of rabeprazole plus 1 g of amoxicillin for 5 days and 20 mg of rabeprazole, 500 mg of clarithromycin, and 500 mg of tinidazole for the remaining 5 days) in 81 patients. All drugs were given twice daily. RESULTS Helicobacter pylori infection was eradicated in 11 of 23 patients (48%) with the A2143G mutation and in 14 of 15 patients (93%) with either A2142G or A2142C strains (difference, 45 percentage points [95% CI, 15 to 65 percentage points]; P = 0.004). The sequential regimen achieved a higher cure rate than triple therapy in A2143G mutate strains (difference, 49 percentage points [CI, 8 to 72 percentage points]; P = 0.024). LIMITATIONS The post hoc substudy design may require further confirmation. Other limitations are the accessibility to the tool and the cost of investigations (70 euros per patient). CONCLUSIONS The A2143G mutation seemed to be associated with a very low eradication rate. The sequential regimen achieved a higher cure rate than standard therapy even in patients with these strains.
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Posteraro P, Branca G, Sanguinetti M, Ranno S, Cammarota G, Rahimi S, De Carlo M, Posteraro B, Fadda G. Rapid detection of clarithromycin resistance in Helicobacter pylori using a PCR-based denaturing HPLC assay. J Antimicrob Chemother 2005; 57:71-8. [PMID: 16284224 DOI: 10.1093/jac/dki406] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
OBJECTIVES We evaluated a new approach for the rapid detection of clarithromycin resistance in Helicobacter pylori, based on PCR and denaturing HPLC (DHPLC). METHODS A 180 bp fragment of the 23S rRNA gene was amplified using DNA from 81 clinical H. pylori isolates (51 isolates were shown to be resistant to clarithromycin by Etest), and, directly, from 101 gastric biopsies from patients with digestive diseases, who were infected with H. pylori as assessed by a 13C-urea breath test, histology and/or culture. DHPLC was used to detect mutations in all the PCR products. RESULTS DHPLC profiles for the 30 susceptible isolates all showed homoduplex peaks; the resistant isolates consistently generated heteroduplex peaks that were easily distinguishable from the wild-type H. pylori reference strain. Sequencing revealed point mutations in all the resistant isolates. Overall, five different mutations were detected. Four of these mutations (A2142G, A2142C, A2143G and T2182C) are known to be associated with clarithromycin resistance; the remaining mutation (C2195T) has not been previously described. This novel single-base substitution was found in combination with the common mutation A2143G. Of the biopsies tested, 25 specimens generated heteroduplexes due to sequence alterations (mutation A2142G, A2142C or A2143G). In one of these specimens, A2143G was found together with the novel mutation T2221C; in another, a mixture of wild-type and mutant (A2143G) sequences was detected. For 20 culture-positive out of the 25 biopsies DHPLC results confirmed the presence of clarithromycin resistance. CONCLUSIONS Our results suggest that the PCR-DHPLC assay is a valid tool for rapid assessment of clarithromycin resistance in H. pylori and that in the future it could be used directly on biopsy specimens, avoiding the need for culture-based methods.
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Affiliation(s)
- Patrizia Posteraro
- Laboratory of Clinical Pathology and Microbiology, Ospedale San Carlo-Istituto Dermopatico dell'Immacolata, Rome, Italy
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Raymond J, Nguyen B, Bergeret M, Dupont C, Kalach N. Heterogeneous susceptibility to metronidazole and clarithromycin of Helicobacter pylori isolates from a single biopsy in adults is confirmed in children. Int J Antimicrob Agents 2005; 26:272-8. [PMID: 16154727 DOI: 10.1016/j.ijantimicag.2005.07.007] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2005] [Accepted: 07/19/2005] [Indexed: 11/22/2022]
Abstract
The aim of this study was to assess the resistance to metronidazole and clarithromycin of individual colonies of Helicobacter pylori from a single biopsy taken from 14 adults and 14 children. The Etest was used to determine the minimum inhibitory concentrations (MICs) of these two antimicrobial drugs for ten individual H. pylori colonies isolated from each initial gastric biopsy culture. We confirmed the heterogeneity in metronidazole and clarithromycin resistance in children as seen in adults before anti-H. pylori treatment. The number of resistant individual colonies ranged from two to nine depending on the subject. All individual colonies from the same biopsy that were resistant to clarithromycin were genetically identical according to randomly amplified polymorphic DNA testing and exhibited the same point mutation according to polymerase chain reaction-restriction fragment length polymorphism.
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Affiliation(s)
- Josette Raymond
- University Paris V, Service Bacteriology, Hôpital Cochin-Saint Vincent de Paul, France.
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Kanai K, Shibayama K, Suzuki S, Wachino JI, Arakawa Y. Growth competition of macrolide-resistant and -susceptible Helicobacter pylori strains. Microbiol Immunol 2005; 48:977-80. [PMID: 15611615 DOI: 10.1111/j.1348-0421.2004.tb03628.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
We examined population dynamics in a mixed culture of clonally related macrolide-resistant and -susceptible Helicobacter pylori strains isolated from a single patient. The resistant strain had a macrolide resistance-conferring A2143G mutation in the 23S rRNA gene. The growth rate of these two strains did not apparently differ when cultured separately. On the other hand, by conducting sequential passage of a mixed culture of the resistant and the susceptible strains, the ratio of the resistant strain to the susceptible strain in the culture typically decreased per passage, indicating that the resistance imposed a significant disadvantage on bacterial fitness in the population.
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Affiliation(s)
- Kyoko Kanai
- Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan
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Simala-Grant JL, Taylor DE. Molecular biology methods for the characterization of Helicobacter pylori infections and their diagnosis. APMIS 2005; 112:886-97. [PMID: 15688524 DOI: 10.1111/j.1600-0463.2004.apm11211-1211.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Helicobacter pylori infects approximately half of the human population; however, the outcome of infection is affected by many factors, including strain and host genotype characteristics and bacterial density within the stomach. Many molecular methods have been developed to provide information with respect to these characteristics. Methods that provide results within 24 h of endoscopy may be used to develop individualized treatment that is more effective, results in fewer side effects, cuts costs,decreases the number of treatment failures and results in the development of fewer antibiotic-resistant H. pylori strains.
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Affiliation(s)
- Joanne L Simala-Grant
- Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada
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Schabereiter-Gurtner C, Hirschl AM, Dragosics B, Hufnagl P, Puz S, Kovách Z, Rotter M, Makristathis A. Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy specimens. J Clin Microbiol 2004; 42:4512-8. [PMID: 15472302 PMCID: PMC522301 DOI: 10.1128/jcm.42.10.4512-4518.2004] [Citation(s) in RCA: 142] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
A biprobe real-time PCR protocol, followed by hybridization melting point analysis, to detect point mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance was established and evaluated in a clinical study. Of 92 patients who underwent endoscopy, 45 were found to be H. pylori infected and invariably were also culture positive. Of the 45 isolates, 11 were shown to be resistant to clarithromycin by E-test. With respect to the detection of H. pylori infection, PCR showed sensitivities of 100% in biopsies and 98% in stool specimens and a specificity of 98% in both biopsy and stool samples. All clarithromycin-sensitive cases were identified as such by PCR in both biopsy and stool samples. Of the cases with a resistant strain, eight were identified as such in stool DNA and nine were identified in biopsy DNA. Failure of PCR to detect the resistant genotype in the biopsy DNA, stool DNA, or both (one case) was associated with mixed populations. In these cases, patients had not been treated for H. pylori infection before, and the sensitive population showed to be present in considerably higher numbers than the resistant population. In five of six cases in which infection with a resistant genotype only was identified by PCR, the patients had received clarithromycin-based eradication therapy in the past. Thus, the assay presented provides a highly accurate noninvasive method to detect H. pylori infection in stool and at the same time allows for culture-independent clarithromycin susceptibility testing.
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Affiliation(s)
- Claudia Schabereiter-Gurtner
- Department of Clinical Microbiology, Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Vienna, Austria
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Abstract
The discovery that most stomach diseases are a consequence of an Helicobacter pylori infection has completely changed the management of stomach diseases. Antibacterials are the treatment of choice in addition to proton pump inhibitors (PPIs) or ranitidine bismuth. We are now faced with the problem of antimicrobial resistance, which is the main cause of treatment failure. H. pylori acquires resistance essentially via point mutations, and today this phenomenon is found with most antibacterials. The most important resistance to consider is that to clarithromycin, since it is the first-choice antibacterial and clarithromycin resistance is highly clinically significant. Quadruple therapy or triple therapies with amoxicillin-metronidazole or tetracycline-metronidazole and a PPI or ranitidine bismuth can then be used despite a possible resistance to metronidazole if the strain is resistant to clarithromycin. Resistance to both clarithromycin and metronidazole may lead to the use of other combinations, i.e. amoxicillin-rifabutin, amoxicillin-levofloxacin or amoxicillin-furazolidone. Resistance to any of these drugs means their use must be avoided. In some instances, it may also be advisable to prescribe amoxicillin as the sole antibacterial, or to use a quadruple therapy with furazolidone instead of metronidazole. Although it is theoretically possible to cure a drug-resistant H. pylori infection, a practical limitation is the availability of the drugs in certain countries. Furthermore, the progressive increase in drug resistance warrants the need for new antibacterials in the near future.
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Affiliation(s)
- Francis Mégraud
- Laboratoire de Bactériologie, Université Victor Segalen Bordeaux 2, Bordeaux, France.
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Affiliation(s)
- F Mégraud
- Laboratoire de Bactériologie, Hôpital Pellegrin, Place Amélie Raba-Léon, 33076 Bordeaux Cedex, France.
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Smith SI, Oyedeji KS, Arigbabu AO, Cantet F, Megraud F, Ojo OO, Uwaifo AO, Otegbayo JA, Ola SO, Coker AO. Comparison of three PCR methods for detection of Helicobacter pylori DNA and detection of cagA gene in gastric biopsy specimens. World J Gastroenterol 2004; 10:1958-60. [PMID: 15222045 PMCID: PMC4572239 DOI: 10.3748/wjg.v10.i13.1958] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To comparatively evaluate PCR and other diagnostic methods (the rapid urease test and/or culture) in order to determine which of the three PCR methods (ureA, glmM and 26-kDa, SSA gene) was most appropriate in the diagnosis of Helicobacter pylori (H pylori ) infection and also to evaluate the detection of a putative virulence marker of H pylori, the cagA gene, by PCR in biopsy specimens.
METHODS: One hundred and eighty-nine biopsy specimens were collected from 63 patients (three biopsies each) undergoing upper gastroduodenal endoscopy for various dyspeptic symptoms. The PCR methods used to detect H pylori DNA directly from biopsies were the glmM, 26-kDa, ureA and then cagA was used to compare the culture technique and CLO for urease with the culture technique being used as the gold standard.
RESULTS: Thirty-five percent of the biopsies were positive for H pylori DNA using the 3 PCR methods, while 68% of these were positive for the cagA gene. Twenty-four percent of the biopsies were negative for H pylori DNA in all PCR methods screened. The remaining 41% were either positive for ureA gene only, glmM only, 26-kDa only, or ureA + glmM, ureA + 26-kDa, glmM + 26-kDa. Out of the 35% positive biopsies, 41% and 82% were positive by culture and CLO respectively, while all negative biopsies were also negative by culture and cagA. Cag A+ infection was also predominantly found in H pylori DNA of the biopsies irrespective of the clinical diagnosis.
CONCLUSION: This method is useful for correctly identifying infections caused by H pylori and can be easily applied in our laboratory for diagnostic purposes.
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Affiliation(s)
- S-I Smith
- Molecular Biology and Biotechnology Division, Nigerian Institute of Medical Research, P.M.B. 2013, Yaba, Lagos, Nigeria.
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Jüttner S, Vieth M, Miehlke S, Schneider-Brachert W, Kirsch C, Pfeuffer T, Lehn N, Stolte M. Reliable detection of macrolide-resistant Helicobacter pylori via fluorescence in situ hybridization in formalin-fixed tissue. Mod Pathol 2004; 17:684-9. [PMID: 15044917 DOI: 10.1038/modpathol.3800098] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Macrolide-resistant Helicobacter (H.) pylori represent an increasing therapeutic problem. Macrolide resistance is usually determined phenotypically in vitro with methods such as E-test or agar dilution test. A prerequisite for those tests, however, is bacterial culture that is not routinely set up in the course of gastroscopy. In contrast, formalin-fixed, paraffin-embedded biopsies are regularly available from patients who have undergone gastroscopy. In such biopsies macrolide-resistant H. pylori can be detected by the genotype-based technique of fluorescence in situ hybridization (FISH). Experience gained by this new method, however, is still extremely limited, especially in formalin-fixed tissue. Therefore, we retrospectively investigated formalin-fixed, paraffin-embedded biopsy specimens by FISH in 104 patients suffering from therapy-resistant H. pylori gastritis. To test the accuracy of FISH, we initially examined specimens from 53 patients for whom results of the E-test were available. Next we analyzed biopsies from another 51 patients that had been selected since phenotypical resistance testing had failed despite documented culturing attempts. In all 104 patients, H. pylori was detected by FISH and could thus be investigated for macrolide resistance. Overall, macrolide-resistant bacteria were found in 71 patients (68.3%). In 49 of 53 patients (92.4%), FISH and E-test returned identical results (no significant discordance according to McNemar's chi(2)-test). Taken together, our study demonstrates that FISH is a highly sensitive and reliable method for detecting macrolide-resistant H. pylori in formalin-fixed, paraffin-embedded biopsy specimens, which represents the routine method of processing tissue obtained upon gastroscopy.
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Affiliation(s)
- Stefan Jüttner
- Institute for Pathology, Klinikum Bayreuth, Bayreuth, Germany
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Hiyama T, Tanaka S, Masuda H, Shima H, Kose K, Tuncel H, Ito M, Kitadai Y, Sumii M, Uemura N, Yoshihara M, Shimamoto F, Haruma K, Chayama K. Prevalence of Helicobacter pylori resistance to clarithromycin and metronidazole determined by 23S ribosomal RNA and rdxA gene analyses in Hiroshima, Japan. J Gastroenterol Hepatol 2003; 18:1202-1207. [PMID: 12974909 DOI: 10.1046/j.1440-1746.2003.03140.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
BACKGROUND AND AIMS Resistance to antibiotics in Helicobacter pylori is increasing and becoming a serious problem in eradication treatment of H. pylori. The prevalence of H. pylori infections that are resistant to clarithromycin, metronidazole, or both were determined in H. pylori isolates in Hiroshima, Japan. METHODS Sixty Japanese patients with H. pylori infection were collected between 1999 and 2000. To detect the resistance to clarithromycin and metronidazole, mutations of the 23S ribosomal RNA (rRNA) and rdxA genes that are responsible for resistance in H. pylori, were examined by direct sequencing analysis. RESULTS Resistance to clarithromycin and metronidazole was detected in 12 (20.0%) and nine (15.0%) of the patients, respectively. Dual resistance to clarithromycin and metronidazole was detected in five (8.3%) patients. CONCLUSION These results indicate that the relatively high prevalence of the dual resistance in H. pylori isolates may need special attention and new therapeutic approaches in Japan.
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Affiliation(s)
- Toru Hiyama
- Health Service Center, Hiroshima University, Higashihiroshima, Japan.
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Bell SJ, Chisholm SA, Owen RJ, Borriello SP, Kamm MA. Evaluation of Helicobacter species in inflammatory bowel disease. Aliment Pharmacol Ther 2003; 18:481-6. [PMID: 12950420 DOI: 10.1046/j.1365-2036.2003.01703.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND Bacteria have been implicated in the pathogenesis of inflammatory bowel disease. Helicobacter species have been shown to cause colitis in animal models and have been identified in human diarrhoeal illness and Crohn's disease. AIM To determine whether Helicobacter species are present in human inflammatory bowel disease tissue. METHODS Thirty patients undergoing colonoscopy for clinical reasons were studied. Nine had Crohn's disease, 11 had ulcerative colitis and 10 had histologically normal colons. Tissue was snap-frozen at -70 degrees C. DNA was extracted and examined by five different polymerase chain reaction (PCR) assays that were either genus or species specific for Helicobacter. RESULTS Analyses of colonic biopsies by two Helicobacter genus-specific PCR assays, two H. pylori-specific assays and a PCR assay designed to amplify fragments of 'H. heilmannii'-like organisms demonstrated that product was not generated by any test. Internal control PCR demonstrated that PCR results for the five assays were not negative due to the presence of residual substances inhibitory to PCR. CONCLUSIONS Helicobacter species were not identified in this study, using multiple PCRs to eliminate the problems of non-specific cross-reaction. This suggests that Helicobacter species do not play a role in the pathogenesis of inflammatory bowel disease.
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Fontana C, Favaro M, Pietroiusti A, Pistoia ES, Galante A, Favalli C. Detection of clarithromycin-resistant Helicobacter pylori in stool samples. J Clin Microbiol 2003; 41:3636-40. [PMID: 12904368 PMCID: PMC179782 DOI: 10.1128/jcm.41.8.3636-3640.2003] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
The recognition of the role of Helicobacter pylori in gastric diseases has led to the widespread use of antibiotics in the eradication of this pathogen. The most advocated therapy, triple therapy, often includes clarithromycin. It is well known that clarithromycin resistance is one of the major causes of eradication failure. The development of a rapid noninvasive technique that could easily be performed on fecal samples and that could also provide information about the antibiotic resistance of this microorganism is therefore advisable. Previous findings have demonstrated that clarithromycin resistance is due to a single point mutation in the 23S rRNA. All the mutations described have been associated with specific restriction sites, namely BsaI (A2143G), MboII (A2142C/G), and HhaI (T2717C). On this basis we have developed a new method, a seminested PCR, allowing screening for clarithromycin resistance of H. pylori directly on stool samples. This method furnished a 783-bp fragment of the 23S rRNA, which was subsequently digested by MboII, BsaI, and HhaI, in order to identify single point mutations associated with clarithromycin resistance. Of a total of 283 stool samples examined, 125 were H. pylori positive and two of them were shown to contain clarithromycin-resistant strains due to the presence of a mutation at position 2717, whereas no PCR products contained mutations at position 2142 or 2143. In order to evaluate the reliability of the new system, we compared the results of restriction analysis of the PCR products with the MICs shown by the H. pylori isolates by culturing gastric biopsies from the same patients.
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Affiliation(s)
- Carla Fontana
- Department of Experimental Medicine and Biochemical Sciences, "Tor Vergata" University of Rome, Italy.
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Chisholm SA, Owen RJ. Development and application of a novel screening PCR assay for direct detection of 'Helicobacter heilmannii'-like organisms in human gastric biopsies in Southeast England. Diagn Microbiol Infect Dis 2003; 46:1-7. [PMID: 12742311 DOI: 10.1016/s0732-8893(02)00547-3] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
A novel PCR assay (HHLO-16) to screen for presence of 'Helicobacter heilmannii'-like organisms (HHLO) direct from gastric biopsies is described. As 'H. heilmannii' is generally uncultivable, diagnosis of infection is reliant on histology; thus prevalence may be underestimated. Analysis of an HHLO histology-positive human gastric biopsy and 15 gastric biopsies from domestic cats demonstrated that the HHLO-16 assay was more sensitive than an alternative available species-specific PCR assay. Further testing of 131 gastric biopsies from dyspeptic patients demonstrated an HHLO prevalence rate of 2.3% in Southeast England. Subsequent combination of the HHLO-16 assay with a H. pylori-specific PCR assay in a multiplex format (HpHh assay), and repeat analysis of the 131 biopsies showed the HpHh assay was as sensitive as each individual test. This novel PCR assay provides simple concomitant testing of dyspeptic patients for both HHLOs and H. pylori, thereby rapidly identifying individuals requiring eradication therapy.
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Affiliation(s)
- Stephanie A Chisholm
- Laboratory of Enteric Pathogens, Central Public Health Laboratory, London NW9 5HT, United Kingdom.
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