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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.

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.

SUBMITTER: Trespalacios AA

PROVIDER: S-EPMC6905078 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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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.

Trespalacios Alba A AA Rimbara Emiko E Otero William W Reddy Rita R Graham David Y DY

Diagnostic microbiology and infectious disease 20141215 4

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 infect ...[more]

PMID: 25600075

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Project description:AimTo investigate the resistance of Helicobacter pylori (H. pylori) to ciprofloxacin (CIP), levofloxacin (LVX) and moxifloxacin (MOX) in the Beijing area and to elucidate the resistance mechanisms.MethodsSeventy-nine H. pylori clinical strains, isolated from patients who had undergone upper gastrointestinal endoscopy in Peking University First Hospital from 2007 to 2009, were tested for their susceptibility to CIP, LVX and MOX using the E-test method. H. pylori strain 26695 was included in the susceptibility testing as a control strain. According to the minimal inhibitory concentration (MIC) values, a strain was classified as resistant to CIP, LVX or MOX when the MIC was > 1 microg/mL. We amplified by polymerase chain reaction (PCR) and sequenced the quinolone resistance-determining regions of the gyrA and gyrB genes from 29 quinolone-resistant and 16 quinolone-susceptible H. pylori strains selected at random.ResultsIn this study, the resistance rates of H. pylori to CIP, LVX or MOX were 55.7% (44/79), and the primary resistance rates were 26.6% (21/79). Patients with secondary resistance had received LVX in previous eradication treatments, but not MOX or CIP. Forty-five strains, including 29 CIP, LVX or MOX-resistant strains (MIC: 1.5-32 microg/mL) and 16 susceptible strains, were selected randomly from the 79 strains and used in PCR analysis. Among these 45 strains, 27 resistant strains had mutations in the gyrA gene, including 11 strains with mutations corresponding to Asp-91 (MIC: 2-32 microg/mL), one of which also had a mutation corresponding to Val-150, and 16 strains had mutations at Asn-87 (MIC: 4-32 microg/mL), three of which also had mutations corresponding to Arg-140 or Val-150. In addition, Arg-140, Val-150 or Ala-97 mutations were separately detected in three susceptible strains. Analysis of the gyrB gene showed that one strain of low resistance had a mutation corresponding to Ser-457 that coexisted with an Asp-91 mutation. There was a significant difference in the occurrence of mutations in the gyrA gene between CIP, LVX and MOX-resistant and -susceptible strains (P < 0.05), but 2 resistant strains were found to possess no quinolone resistance-determining region mutations.ConclusionResistance is primarily mediated through point mutations in gyrA. Whether other mechanisms are responsible for resistance in strains without mutations in the QRDR should be detected.

Project description:To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR).The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion.The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin.The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

Dataset Information

Improved allele-specific PCR assays for detection of clarithromycin and fluoroquinolone resistant of Helicobacter pylori in gastric biopsies: identification of N87I mutation in GyrA.

Publications

Improved allele-specific PCR assays for detection of clarithromycin and fluoroquinolone resistant of Helicobacter pylori in gastric biopsies: identification of N87I mutation in GyrA.

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