Dataset Information

Microarray-Based Detection and Clinical Evaluation for Helicobacter pylori Resistance to Clarithromycin or Levofloxacin and the Genotype of CYP2C19 in 1083 Patients.

ABSTRACT: Background. Helicobacter pylori (H. pylori) is one of the most frequent and persistent bacterial infections that affect nearly half of the world's population. Antibiotic resistance is a constantly evolving process and local surveillance of antibiotic resistance is warranted to guide clinicians in their choice of therapy. The aim of this study was to establish a microarray-based detection to identify H. pylori infection, clarithromycin and levofloxacin susceptibility, and CYP2C19 genetic polymorphism and guide to potential choice of proton pump inhibitor (PPI), antibiotic administration for tailored H. pylori eradication therapy. Methods. By analyzing the sequence of human genomic CYP2C19?2 and CYP2C19?3 and mutations within the 23S rRNA and gyrA gene regions conferring clarithromycin and levofloxacin resistance, respectively, we developed a microarray for individual therapy detection of H. pylori infection. Plasmids were established as positive or limit of detection (LOD) reference materials. The specificity and sensitivity of the microarray had been performed. And a total of 1083 gastric biopsy samples were tested and the Kappa value had been calculated between the array and Sanger sequencing. We also analyzed the resistance to clarithromycin and levofloxacin in China, as well as the CYP2C19 polymorphisms. Results. The LOD of detecting H. pylori was 103 CFU/mL and human genome DNA was 2?ng/?L. The detection results of 1083 gastric biopsy samples showed that 691 (63.80%) were H. pylori positive, of which 266 (38.49%) were resistant to clarithromycin, 192 (27.79%) were resistant to levofloxacin, and 61 (8.83%) were resistant to both of them. For the type of CYP2C19 polymorphism, 412 (38.04%) were homozygous fast type (HomEM), 574 (53%) were heterozygous EM (HetEM), and 97 (8.96%) were poor metabolizer (PM). Conclusions. The proposed microarray-based detection has high specificity, sensitivity, and reproducibility for detecting the resistance of clarithromycin or levofloxacin as well as CYP2C19 polymorphism, which may help to improve the clinical eradication rate of H. pylori.

SUBMITTER: Song Y

PROVIDER: S-EPMC6151853 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Microarray-Based Detection and Clinical Evaluation for Helicobacter pylori Resistance to Clarithromycin or Levofloxacin and the Genotype of CYP2C19 in 1083 Patients.

Song Yi Y Dou Fengna F Zhou Zhe Z Yang Ningmin N Zhong Jing J Pan Jie J Liu Qiqi Q Zhang Jianzhong J Wang Shengqi S

BioMed research international 20180910

Background. Helicobacter pylori (H. pylori) is one of the most frequent and persistent bacterial infections that affect nearly half of the world's population. Antibiotic resistance is a constantly evolving process and local surveillance of antibiotic resistance is warranted to guide clinicians in their choice of therapy. The aim of this study was to establish a microarray-based detection to identify H. pylori infection, clarithromycin and levofloxacin susceptibility, and CYP ...[more]

PMID: 30276203

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Project description:IntroductionUnderstanding drug resistance is important in drug selection for Helicobacter pylori (H. pylori) eradication, and drug resistance data are lacking in Beijing.PurposeThis cross-sectional study aimed to isolate H. pylori from patients with gastroduodenal diseases and to analyze drug resistance to clarithromycin (CLA) and levofloxacin (LEV), which are used frequently in China.Patients and methodsOne hundred and seventy-six patients with gastroduodenal diseases undergoing gastroduodenoscopy were selected by convenient sampling. Gastric mucosa samples were cultured and sub-cultured using a new medium broth. Active H. pylori strains were confirmed by microscopy observation as Gram-negative curved bacilli with positive test results for urease, oxidase, and catalase, and H. pylori 16S rRNA amplification by polymerase chain reaction (PCR). CLA and LEV resistance was identified by minimum inhibitory concentration (MIC) tests and sequencing of 23S rRNA, gyrA, and gyrB genes.ResultsFrom the 176 clinical samples, 112 (112/176, 63.6%) were confirmed with H. pylori infection and 65 (65/176, 36.9%) active H. pylori strains were obtained and further confirmed by MIC assay. Overall, the rates of CLA-resistant and LEV-resistant mutations in the 112 samples were 50.9% and 33.0%, respectively. Mutation related to CLA resistance was A2143G in the 23S rRNA gene and mutations associated with LEV resistance were N87K, D91G, and D91Y in the gyrA gene. Of 112 samples, 22 (19.6%) presented dual resistance to CLA and LEV. Resistance of the H. pylori strains to CLA (r=0.846, P<0.001) and LEV (r=0.936, P<0.001) had a strong correlation in phenotypic and genotypic level.ConclusionThe results indicated that resistance of CLA and LEV is severe among patients with gastroduodenitis. A good consistency could be found as to drug resistance between genotypic or phenotypic assay, suggested extending the detection of H. pylori drug resistance from the MIC method to a genotypic assay.

Project description:BackgroundDue to increased prevalence of H. pylori antimicrobial resistance worldwide and more importantly the resistance patterns vary between different geographical regions, it is important to survey local H. pylori antibiotic resistance profile to provide physicians with more informed drug choices to better treat H. pylori infection. To our knowledge, this is the first study to examine the prevalence of antimicrobial resistance of H. pylori in Karnataka state of South India.ResultsA total of 113 H. pylori strains were isolated from gastric biopsies and tested: 81.4% were resistant to metronidazole, 54.9% were resistant to levofloxacin, 20.4% were resistant to clarithromycin, 5.3% were resistant to tetracycline and 7.1% were resistant to amoxicillin. Multidrug resistance was detected in 59.3% of total isolated strains, among which 86.6% were resistant to at least both metronidazole and levofloxacin. In this study, 38 out of 113 H. pylori strains had been whole-genome sequenced. Based on the draft genomes, RdxA and/or FrxA inactivation mutations were found to present in 75% of metronidazole-resistant strains. Clarithromycin-resistant strains had mainly A2143G and G2224A mutations in the 23 rRNA gene. While 87.1% levofloxacin-resistant strains had amino acid substitution mutations occurring predominantly at N87 and D91 in GyrA, novel mutations in the same protein including an insertion of five amino acid residues (QDNSV), immediately after the start codon, and a substitution mutation at R295 were identified.ConclusionHigh primary resistance to metronidazole and levofloxacin, and a modest occurrence of clarithromycin resistance were revealed in H. pylori strains isolated from Karnataka patients. Therefore metronidazole-, levofloxacin- and clarithromycin-based triple therapies are not suitable as first-line treatment in Karnataka. Both amoxicillin and tetracycline can still be used to eradicate H. pylori infection in this region. We also revealed novel mutations in GyrA protein that possibly contribute to H. pylori resistance in levofloxacin, which merit further investigations.

Project description:Aims and objectivesMore than half of the world's population is infected with Helicobacter pylori, which can cause chronic gastritis. WHO has regarded clarithromycin-resistant H. pylori as a high priority pathogen. Hence, accurate diagnosis and detection of clarithromycin- and levofloxacin-resistant H. pylori strains is essential for proper management of infection. The objective of this study was to develop and optimize multiplex quantitative PCR assay for detection of mutations associated with clarithromycin and levofloxacin resistance in H. pylori directly from the gastric biopsies.Materials and methodsSpecific primers and probes were designed to amplify ureA and mutations in 23S rRNA and gyrA genes. Singleplex and triplex qPCR assays were optimized and the assay's sensitivities and specificities were determined. The optimized multiplex qPCR assay was performed on 571 gastric biopsies.ResultsIn this study, 14.7% (84/571) of the gastric biopsies were positive for H. pylori by conventional methods and 23.8% (136/571) were positive by the ureA-qPCR with 96.4% sensitivity and 88.5% specificity, while the +LR and -LR were 8.72 and 0.04, respectively. The ureA-positive samples (n=136) were subjected to multiplex qPCR which detected A2142G and A2143G mutations in the 23S rRNA gene (20.6%, 28/136) conferring clarithromycin resistance and gyrA mutations N87K, N87I, D91N, and D91Y (11.8%, 16/136) leading to levofloxacin resistance. The sensitivity and specificity of qPCR of 23S rRNA gene were 100% and 98.7%, respectively, while 100% and 99.8% for qPCR of gyrA, respectively.ConclusionThe effectiveness of this qPCR is that it is sensitive in detecting low bacterial load and will help in timely detection of clarithromycin- and levofloxacin-resistant strains, especially in case of mixed infections. Since it is culture independent, it can inform clinicians about antibiotics to be included in the first-line therapy, thereby improving the management of H. pylori infection at a much greater pace.

Dataset Information

Microarray-Based Detection and Clinical Evaluation for Helicobacter pylori Resistance to Clarithromycin or Levofloxacin and the Genotype of CYP2C19 in 1083 Patients.

Publications

Microarray-Based Detection and Clinical Evaluation for <i>Helicobacter pylori</i> Resistance to Clarithromycin or Levofloxacin and the Genotype of CYP2C19 in 1083 Patients.

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