Dataset Information

Peptide Nucleic Acid Probe-Based Analysis as a New Detection Method for Clarithromycin Resistance in Helicobacter pylori.

ABSTRACT:

Background/aims

Helicobacter pylori eradication rates are decreasing because of increases in clarithromycin resistance. Thus, finding an easy and accurate method of detecting clarithromycin resistance is important.

Methods

We evaluated 70 H. pylori isolates from Korean patients. Dual-labeled peptide nucleic acid (PNA) probes were designed to detect resistance associated with point mutations in 23S ribosomal ribonucleic acid gene domain V (A2142G, A2143G, and T2182C). Data were analyzed by probe-based fluorescence melting curve analysis based on probe-target dissociation temperatures and compared with Sanger sequencing.

Results

Among 70 H. pylori isolates, 0, 16, and 58 isolates contained A2142G, A2143G, and T2182C mutations, respectively. PNA probe-based analysis exhibited 100.0% positive predictive values for A2142G and A2143G and a 98.3% positive predictive value for T2182C. PNA probe-based analysis results correlated with 98.6% of Sanger sequencing results (?-value=0.990; standard error, 0.010).

Conclusions

H. pylori clarithromycin resistance can be easily and accurately assessed by dual-labeled PNA probe-based melting curve analysis if probes are used based on the appropriate resistance-related mutations. This method is fast, simple, accurate, and adaptable for clinical samples. It may help clinicians choose a precise eradication regimen.

SUBMITTER: Jung DH

PROVIDER: S-EPMC6254629 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Publications

Peptide Nucleic Acid Probe-Based Analysis as a New Detection Method for Clarithromycin Resistance in <i>Helicobacter pylori</i>.

Jung Da Hyun DH Kim Jie-Hyun JH Jeong Su Jin SJ Park Soon Young SY Kang Il-Mo IM Lee Kyoung Hwa KH Song Young Goo YG

Gut and liver 20181101 6

<h4>Background/aims</h4><i>Helicobacter pylori</i> eradication rates are decreasing because of increases in clarithromycin resistance. Thus, finding an easy and accurate method of detecting clarithromycin resistance is important.<h4>Methods</h4>We evaluated 70 <i>H. pylori</i> isolates from Korean patients. Dual-labeled peptide nucleic acid (PNA) probes were designed to detect resistance associated with point mutations in 23S ribosomal ribonucleic acid gene domain V (A2142G, A2143G, and T2182C). ...[more]

PMID: 30037168

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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.