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

Jung, Da Hyun; Kim, Joo Hang; Jeong, Su Jin; Park, Soon Young; Kang, Il‐Mo; Lee, Kyoung Hwa; Song, Young Goo

doi:10.5009/gnl18111

Cited by 17 publications

(12 citation statements)

References 36 publications

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“…The critical essence of these two mutations in incurring resistance on susceptible H pylori strains has also been proven by site‐directed mutagenesis 57 . Again, in agreement with others, 58‐60 in our limited number of resistant strains, we have also found the mentioned mutations, as the major (8/11) cause of CLA resistance. We have also detected other mutations (T2182C, T2656A, and G2569C), which were outside of the drug‐interacting region and thus did not undergo our molecular docking studies.…”

Section: Discussionsupporting

confidence: 90%

The outward shift of clarithromycin binding to the ribosome in mutantHelicobacter pyloristrains

et al. 2020

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Objectives Disruption of protein synthesis, by drug‐mediated restriction of the ribosomal nascent peptide exit tunnel (NPET), may inhibit bacterial growth. Here, we have studied the secondary and tertiary structures of domain V of the 23S rRNA in the wild‐type and mutant (resistant) H. pylori strains and their mechanisms of interaction with clarithromycin (CLA). Methods H pylori strains, isolated from cultured gastric biopsies, underwent CLA susceptibility testing by E test, followed by PCR amplification and sequencing of domain V of 23S rRNA. The homology model of this domain in H pylori, in complex with L4 and L22 accessory proteins, was determined based on the E. coli ribosome 3D structure. The interactions between CLA and 23S rRNA complex were determined by molecular docking studies. Results Of the 70 H pylori strains, isolated from 200 dyspeptic patients, 11 (16%) were CLA‐resistant. DNA sequencing identified categories with no (A), A2142G (B), and A2143G (C) mutations. Docking studies of our homology model of 23S rRNA complex with CLA showed deviated positions for categories B and C, in reference to category A, with 12.19 Å and 7.92 Å RMSD values, respectively. In both mutant categories, CLA lost its interactions at positions 2142 and 2587 and gained two new bonds with the L4 accessory protein. Conclusion Our data suggest that, in mutant H pylori strains, once the nucleotides at positions 2142 and 2587 are detached from the drug, CLA interacts with and is peeled back by the L4 accessory protein, removing the drug‐imposed spatial restriction of the NPET.

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Section: Discussionsupporting

confidence: 90%

The outward shift of clarithromycin binding to the ribosome in mutantHelicobacter pyloristrains

et al. 2020

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“…Moreover, PNA probe‐based qPCR allows easy interpretation of mutation type using a graph of the melting peak. It has recently been reported that the same PNA probe‐based qPCR kit used in this study shows 100% positive predictive value and high κ‐value for detecting the presence and mutation status of H pylori in cultured H pylori samples isolated from Korean patients when compared to conventional PCR with sequencing analysis . Taken together, the PNA probe‐based qPCR method is a simple and accurate method for detection of H pylori in gastric biopsies.…”

Section: Discussionsupporting

confidence: 51%

Detection of Helicobacter pylori with clarithromycin resistance‐associated mutations using peptide nucleic acid probe‐based melting point analysis

et al. 2019

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Background: Detection of Helicobacter pylori in gastric biopsy is important for appropriate treatment and prevention of gastric carcinoma and lymphoma. A novel peptide nucleic acid probe (PNA)-based real-time polymerase chain reaction (PCR) method was developed for detection of H pylori and A2142G/A2143G mutation of the 23S rRNA gene, which is associated with clarithromycin resistance. Methods: To evaluate the performance of the PNA probe-based PCR method, a total of 409 gastric biopsy samples were analyzed by PNA probe-based PCR and compared with other H pylori detection methods, including hematoxylin and eosin (HE) and Warthin-Starry (WS) staining, immunohistochemistry (IHC). A2142G/A2143G mutation of the 23S rRNA gene was tested by dual priming oligonucleotide (DPO)based PCR and Sanger sequencing to evaluate PNA probe-based PCR.Results: Among 271 cases that were positive for H pylori on IHC which was considered as a standard method, 264 cases (97.4%) and 259 cases (95.6%) were positively detected by HE/WS and PNA probe-based qPCR, respectively. Of 100 H pyloripositive patients tested by IHC, H pylori was detected in 93 cases (93.0%) by PNA probe-based PCR, 86 cases (86.0%) by DPO-based PCR, and 93 cases (93.0%) by conventional PCR. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of PNA probe-based qPCR were 93.0%, 94.9%, 93.9%, 94.9%, and 93.0%, respectively, which were all higher than those of DPO-based PCR.When Sanger sequencing was determined as a standard method to detect A2142G/ A2143G mutations, the sensitivity of the PNA-and DPO-based methods was 100% and 94.4%, respectively, and the specificity was 100% for both methods.Conclusion: PNA probe-based qPCR is an appropriate method for detecting H pylori and the clarithromycin resistance-associated mutation type. K E Y W O R D Sclarithromycin resistance, gastric biopsy, Helicobacter pylori diagnosis, immunohistochemistry, peptide nucleic acid probe, real-time polymerase chain reaction

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“…The quasi-species development of H. pylori in a single host might result in treatment failure even after a tailored eradication strategy based on the presence of a 23S ribosomal RNA point mutation[28]. Second, a DPO-PCR-based evaluation is limited to detecting mutations of A2142G and A2143G in 23S rRNA, and other mutations, such as the A2144G or A2142C mutation of H. pylori , cannot be detected[20,30].…”

Section: Discussionmentioning

confidence: 99%

Tailored eradication vs empirical bismuth-containing quadruple therapy for first-line Helicobacter pylori eradication: A comparative, open trial

Choi

Chung

Park

et al. 2019

WJG

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BACKGROUNDFew studies have compared the efficacy and safety profile of a tailored eradication (TR) strategy based on the presence of a 23S ribosomal RNA point mutation with those of empirical bismuth-based quadruple therapy (EBQT) for first-line eradication of Helicobacter pylori (H. pylori) in Korean patients.AIMTo compare the efficacy and safety of a TR strategy and those of EBQT regimen as first-line eradication therapy for H. pylori.METHODSThis is an open-label, comparative study in which we prospectively enrolled patients over 18 years of age with H. pylori infection and retrospectively reviewed their data. H. pylori-positive patients diagnosed by rapid urease test, Giemsa staining, or dual priming oligonucleotide polymerase chain reaction (DPO-PCR) were enrolled from May 2016 to September 2018 at Gil Medical Center. Patients with H. pylori infection received either a TR regimen or the EBQT regimen. In the tailored therapy group that underwent DPO-PCR testing, patients with A2142G and/or A2143G point mutations were treated with a bismuth-containing quadruple regimen. The eradication rate, patient-reported side effect rate, and H. pylori eradication success rate were evaluated and compared between the groups.RESULTSA total of 150 patients were assigned to the TR (n = 50) or EBQT group (n = 100). The first-line eradication rate of H. pylori did not differ between the groups (96.0% vs 95.7%, P = 0.9). The rate of eradication-related side effects for TR was 12.0%, which differed significantly from that of EBQT (43.0%) for first-line treatment (P < 0.001).CONCLUSIONDPO-PCR-based TR for H. pylori eradication may be equally efficacious, with less treatment-related complications, compared to EBQT in Korea, where clarithromycin resistance is high.

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

Cited by 17 publications

References 36 publications

The outward shift of clarithromycin binding to the ribosome in mutantHelicobacter pyloristrains

The outward shift of clarithromycin binding to the ribosome in mutantHelicobacter pyloristrains

Detection of Helicobacter pylori with clarithromycin resistance‐associated mutations using peptide nucleic acid probe‐based melting point analysis

Tailored eradication vs empirical bismuth-containing quadruple therapy for first-line Helicobacter pylori eradication: A comparative, open trial

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