Article Search
검색
검색 팝업 닫기

Metrics

Help

  • 1. Aims and Scope

    Gut and Liver is an international journal of gastroenterology, focusing on the gastrointestinal tract, liver, biliary tree, pancreas, motility, and neurogastroenterology. Gut atnd Liver delivers up-to-date, authoritative papers on both clinical and research-based topics in gastroenterology. The Journal publishes original articles, case reports, brief communications, letters to the editor and invited review articles in the field of gastroenterology. The Journal is operated by internationally renowned editorial boards and designed to provide a global opportunity to promote academic developments in the field of gastroenterology and hepatology. +MORE

  • 2. Editorial Board

    Editor-in-Chief + MORE

    Editor-in-Chief
    Yong Chan Lee Professor of Medicine
    Director, Gastrointestinal Research Laboratory
    Veterans Affairs Medical Center, Univ. California San Francisco
    San Francisco, USA

    Deputy Editor

    Deputy Editor
    Jong Pil Im Seoul National University College of Medicine, Seoul, Korea
    Robert S. Bresalier University of Texas M. D. Anderson Cancer Center, Houston, USA
    Steven H. Itzkowitz Mount Sinai Medical Center, NY, USA
  • 3. Editorial Office
  • 4. Articles
  • 5. Instructions for Authors
  • 6. File Download (PDF version)
  • 7. Ethical Standards
  • 8. Peer Review

    All papers submitted to Gut and Liver are reviewed by the editorial team before being sent out for an external peer review to rule out papers that have low priority, insufficient originality, scientific flaws, or the absence of a message of importance to the readers of the Journal. A decision about these papers will usually be made within two or three weeks.
    The remaining articles are usually sent to two reviewers. It would be very helpful if you could suggest a selection of reviewers and include their contact details. We may not always use the reviewers you recommend, but suggesting reviewers will make our reviewer database much richer; in the end, everyone will benefit. We reserve the right to return manuscripts in which no reviewers are suggested.

    The final responsibility for the decision to accept or reject lies with the editors. In many cases, papers may be rejected despite favorable reviews because of editorial policy or a lack of space. The editor retains the right to determine publication priorities, the style of the paper, and to request, if necessary, that the material submitted be shortened for publication.

Search

Search

Year

to

Article Type

Online first

Split Viewer

Online first

Real-Time Polymerase Chain Reaction for the Detection of Helicobacter pylori and Clarithromycin Resistance

Jin Hee Noh1 , Ji Yong Ahn1 , Jene Choi2 , Young Soo Park2 , Hee Kyong Na1 , Jeong Hoon Lee1 , Kee Wook Jung1 , Do Hoon Kim1 , Kee Don Choi1 , Ho June Song1 , Gin Hyug Lee1 , Hwoon-Yong Jung1 , Jung Mogg Kim3

Departments of 1Gastroenterology and 2Pathology, Asan Medical Center, University of Ulsan College of Medicine, and 3Department of Microbiology, Hanyang University College of Medicine, Seoul, Korea

Correspondence to: Ji Yong Ahn
ORCID https://orcid.org/0000-0002-0030-3744
E-mail ji110@hanmail.net

Received: February 24, 2022; Revised: March 30, 2022; Accepted: April 14, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Gut Liver

Published online July 20, 2022

Copyright © Gut and Liver.

Background/Aims: Real-time polymerase chain reaction (RT-PCR) is a fast and simple method for the simultaneous detection of clarithromycin (CLR) resistance and Helicobacter pylori. We evaluated the effectiveness of RT-PCR compared to that of the rapid urease test (RUT) and assessed its value in verifying CLR resistance.
Methods: A total of 70 specimens with confirmed H. pylori infection in culture were enrolled and analyzed in this prospective study. All specimens were subjected to RT-PCR assay using fluorescence melting peak signals to detect H. pylori infection and CLR resistances caused by either A2142G or A2143G mutations in the 23S ribosomal RNA gene (23S rRNA). The results were compared to those of RUT and antimicrobial susceptibility culturing tests to investigate the efficacy of RT-PCR.
Results: Among the 70 specimens analyzed, the positivity rate was 97.1% (68/70) with RT-PCR and 82.9% (58/70) with RUT. CLR resistance (minimum inhibitory concentration >1.0 μg/mL) was confirmed in 18.6% (13/70), and fluorescence melting curve analysis showed that 84.6% (11/13) had point mutations in 23S rRNA. Ten specimens had only A2143G mutation, and one specimen contained both A2142G and A2143G mutations.
Conclusions: RT-PCR assay was found to be more efficient than RUT in detecting H. pylori infection and could effectively verify CLR resistance compared to the antimicrobial susceptibility culturing test. Considering the high sensitivity and accessibility of RT-PCR method, it could be used to easily detect CLR-resistant H. pylori, thus helping clinicians select suitable treatment regimen and improve the eradication rate.

Keywords: Clarithromycin resistance, Helicobacter pylori, Culture, Rapid urease test, Real-time polymerase chain reaction

Helicobacter pylori is a Gram-negative, spiral-shaped bacterium that colonizes the gastric mucosa and may cause atrophic gastritis, peptic ulcers, gastric cancer, and mucosal-associated lymphoid tissue lymphoma. Therefore, eradication of H. pylori is important not only for patients infected with the disease but also for asymptomatic, healthy individuals. With a global increase in antibiotic resistance, the success rate of H. pylori eradication is decreasing.1-3 The main cause of treatment failure has been reported as clarithromycin (CLR) resistance, and among Korean patients, CLR resistance has gradually increased from 13.7% in 2003 to 16.7% in 2006 and 23.7% in 2012.4 Thus, it is important to detect CLR resistance and select an appropriate treatment regimen to improve the H. pylori eradication rate.

The rapid urease test (RUT) is the most common method for detecting H. pylori infection. It yields results quickly, has high sensitivity and specificity, and is economic and simple to use. However, it requires a high density of bacteria and cannot detect antibiotic resistance.5 Culture and antibiotic susceptibility tests can confirm antibiotic resistance regardless of genetic mutations, but culturing bacterial takes at least 5 to 7 days. Additionally, because the culture conditions and composition environments may vary depending on the institution and are difficult to construct, the culture success rate ranges from 50% to 80%.6 However, the real-time polymerase chain reaction (RT-PCR) assay is a fast and simple method for the simultaneous detection of CLR resistance and H. pylori.7 Although it is a sensitive and specific method for detecting H. pylori infection in gastric tissue samples, there is a lack of studies comparing the RT-PCR assay, RUT, and antibiotic susceptibility test using conventional culture.

Therefore, we evaluated whether the RT-PCR assay is more efficient than RUT for detecting H. pylori and whether it is appropriate for identifying CLR resistance with the associated mutations.

1. Study design and population

This prospective study was conducted between December 2019 and December 2020 at Asan Medical Center, Seoul, Korea. This study was approved by the Institutional Review Board of Asan Medical Center (IRB number: 2020-0082) and conducted in accordance with the standards of the Declaration of Helsinki. The patients, who were between 19 and 80 years old and had no history of H. pylori eradication, were enrolled after providing written informed consent. Patients with a history of gastric surgery, hematologic disease with bleeding tendency, or antibiotics administration within the past month were excluded.

All patients underwent serum H. pylori immunoglobulin G (IgG) testing and esophagogastroduodenoscopy with pairs of mucosal biopsies from gastric antrum and corpus for each RUT and culture with antimicrobial susceptibility tests. Of these, the specimens from patients who were positive for either antrum or body on RUT were used for the RT-PCR assay with U-TOPTM HPy & ClaR Detection Kit (SeaSun Biomaterials, Daejeon, Korea). Finally, the 70 specimens that were identified as having H. pylori infection in culture, which was considered a confident method, were analyzed to investigate the H. pylori detection of RT-PCR assay and RUT, as well as the presence of CLR resistances by detecting A2142G or A2143G mutations in the 23S ribosomal RNA gene (23S rRNA) (Supplementary Fig. 1).

2. H. pylori culture

The tissues obtained from gastric antrum and corpus during esophagogastroduodenoscopy were first placed in a sterile Eppendorf tube and then placed in a vacuum bottle containing dry ice. The Eppendorf tubes were stored in a –80°C deep freezer and the tissues were allowed to thaw at room temperature before analysis. The specimens were inoculated onto an H. pylori isolation medium (Brucella broth agar supplemented with 5% sheep blood and containing 10 μg/L vancomycin, 5 μg/L trimethoprim, 5 μg/L amphotericin B, and 2.5 IU polymyxin B) and incubated at 37°C under microaerophilic conditions (5% O2, 10% CO2, and 85% N2) for 5 to 7 days. Suspected H. pylori colonies were identified based on morphology, Gram staining, and urease positive reaction.

The minimum inhibitory concentrations (MICs) of antibiotics were determined using the serial 2-fold agar dilution method as described previously.8 Bacteria were sub-cultured for 48 hours on Mueller–Hinton agar supplemented with 5% defibrinated sheep blood. The bacterial suspension was adjusted to 1×107 colony-forming units and inoculated to each antibiotic-containing agar dilution plate followed by incubation for 3 days at 37°C under microaerophilic conditions. The MIC of antibiotics was evaluated after 72 hours. A standard H. pylori strain (ATCC 43504) was used as the control. Resistance breakpoint for CLR was set as >1 μg/mL.9

3. RT-PCR for detection of H. pylori infection and CLR resistance

According to the manufacturer's instruction (QIAamp DNA Tissue Kit; Qiagen, Hilden, Germany), the genomic DNA was extracted from the leftover biopsied samples after performing the RUT. The quality and quantity of the genomic DNA samples were evaluated using a Nanodrop spectrophotometers (Thermo Fisher, Waltham, MA, USA), with a >15 ng/μL cutoff value of appropriate bacterial concentration for the test. RT-PCR for detection of H. pylori and CLR resistances was performed with the U-TOPTM HPy & ClaR Detection Kit (SeaSun Biomaterials) using the CFX96 Real-Time PCR detection system V1.6 (Bio-Rad, Hercules, CA, USA). Specific primers were used to amplify the H. pylori genes. The polymerase nucleic acid probe-based qPCR method was used for analyzing the mutations status of H. pylori. Data were analyzed using the SS AnalyzerII V1.0 (SeaSun Biomaterials, Seoul, Korea) software. Infection and CLR resistance were differentiated by the fluorescence melting peak signal (Tm) (Fig. 1A). For cases with mixed infection of wild type and mutant H. pylori, melt curve analysis was further performed to distinguish two different peaks according to each percentage, and the presence of point mutation was differentiated using the gradient difference (Fig. 1B and C).

Figure 1.Representative results of internal positive control (IPC), Helicobacter pylori-positive, A2142G mutation, and A2143G mutation using U-TOPTM HPy & ClaR Detection Kit. (A) Examples of melting peak signal (Tm) with standard value. (B) Tm results according to the percentage of wild type and mutant (A2142G and A2143G point mutations) in mixed-infection samples, and (C) examples with different percentage.

4. Statistical analysis

Descriptive statistics for the categorical variables were summarized as proportions, and the continuous variables were summarized using the medians (interquartile range). The McNemar test was applied to calculate the diagnostic sensitivity of each test. The sensitivity (%) was calculated as follows: 100×true positives/(true positives+false negatives). The true positives and false negatives were determined depending on whether the H. pylori infection was confirmed by culture. The specificity (%) of the method was calculated as follows: 100×true negatives/(false positives+true negatives), where true negatives and false positives were defined according to H. pylori infection determined by combining the results of culture and H. pylori IgG antibody test. All statistical analyses were conducted using SPSS version 24 (IBM Corp., Armonk, NY, USA).

1. Clinical characteristics and diagnostic tests

A total of 70 specimens from 39 patients were included in this study. The median age was 62 years (interquartile range, 58 to 70 years), and 87% of the patients (34/39) were male. Indications of test were 56% (22/39) of dysplasia and 44% (17/39) of early gastric cancer. The RUT results showed 72% (23/32) antrum positivity and 92% (35/38) body positivity, whereas the RT-PCR results showed 94% (30/32) antrum positivity and 100% (38/38) body positivity. The results of H. pylori IgG antibody test, RUT, and RT-PCR of the 70 samples are summarized in Supplementary Table 1.

2. Comparison of H. pylori detection rate using RUT and RT-PCR

Among the 70 specimens, RT-PCR and RUT assays showed sensitivities of 97.1% (68/70) and 82.9% (58/70) (Fig. 2), and specificities of 100% (8/8) and 62.5% (5/8), respectively. Table 1 shows the accordance of each H. pylori diagnostic test according to the stomach location, namely, the antrum and body. The RUT and RT-PCR assay showed accordance of 70% (21/30) for the antrum and 92% (35/38) for the body. The RUT and culture represented 72% (23/32) and 92% (35/38) of accordance for the antrum and body, respectively. In the case of RT-PCR and culture, the accordance was 94% (30/32) for the antrum and 100% (38/38) for the body. Consequently, the body specimens showed higher accordance between the two tests than the antrum specimens.

Table 1. Accordance among RUT, RT-PCR, and Culture for Helicobacter pylori Diagnosis According to Its Location in the Stomach of Culture-Positive Specimens (n=70)

VariableRUTRT-PCR
AntrumBodyAntrumBody
Culture (n=70)Antrum (n=32)23 (72)30 (94)
Body (n=38)35 (92)38 (100)
RT-PCR (n=68)Antrum (n=30)21 (70)**
Body (n=38)35 (92)**

Data are presented as number (%).

RUT, rapid urease test; RT-PCR, real-time polymerase chain reaction.

*Comparisons of the same test (RT-PCR).


Figure 2.Comparison of Helicobacter pylori detection rates between the rapid urease test (RUT) and real-time polymerase chain reaction (RT-PCR) assay in culture-positive specimens.

3. CLR resistance by culture and RT-PCR

Fig. 3 shows the CLR resistance in culture and the proportions of point mutations. Among the 70 specimens, 13 (18.6%) were CLR resistant (MIC >1.0 μg/mL) and 57 (82.3%) were CLR susceptible. Of the CLR-resistant specimens, 11 (84.6%) had 23S ribosomal RNA point mutations, detected using fluorescence melting curve analysis. The A2143G mutation was observed in 10 specimens, and notably, one specimen showed A2142G and A2143G double mutations. The MIC of CLR and the mutation types of each specimen are summarized in Table 2.

Table 2. MIC of CLR and Mutation Types Detected Using RT-PCR Assay on CLR-Resistant Specimens

Specimen numberCLR MIC, mg/LMutation type
1>128A2142G, A2143G
2>128A2143G
364A2143G
464A2143G
532A2143G
616A2143G
716A2143G
816A2143G
916A2143G
1016A2143G
118A2143G
128-
138-

MIC, minimum inhibitory concentration; CLR, clarithromycin; RT-PCR, real-time polymerase chain reaction.

RT-PCR was performed using the U-TOPTM HPy & ClaR Detection Kit (SeaSun Biomaterials).


Figure 3.Clarithromycin (CLR) resistance and proportion of point mutations (A2142G and A2143G) confirmed using agar dilution test and real-time polymerase chain reaction (RT-PCR).

Although several PCR-based H. pylori detection methods with high sensitivity and specificity have recently been reported, few studies have clinically focused on the comparison between PCR and other methods such as RUT and standard susceptibility culturing tests. In this study, we investigated whether the RT-PCR assay is more efficient than the RUT for detecting H. pylori and whether it is more appropriate for identifying CLR resistance compared to the susceptibility culturing test. Among the 70 specimens with confirmed H. pylori infection in culture, 97.1% and 82.9% were positively confirmed using RT-PCR and RUT, respectively. The CLR resistance in culture was confirmed in 18.6% of the specimens, and 84.6% of them had A2142G and A2143G mutations in the 23S rRNA gene, as detected using the fluorescence melting curve analysis. Additionally, H. pylori IgG antibody test was performed using all specimens, of which, four were found to be negative. The sensitivity of H. pylori IgG antibody test was 94.3% (66/70), similar to those reported in previous studies.10,11

According to previous studies, the results of H. pylori detection and CLR resistance of RT-PCR-based assays were similar to those of the commercialized DPO-PCR (Seeplex ClaR-H. pylori ACE Detection; Seegene Institute of Life Sciences, Seoul, Korea) and conventional PCR.12,13 RT-PCR-based assay is a simple and accurate method for detecting H. pylori and mutation types of CLR resistance that can be analyzed using a small formalin-fixed, paraffin-embedded specimen after amplification. As it is a one-step analysis, it can avoid cross-contamination that can occur during electrophoresis. Moreover, RT-PCR allows easy and accurate interpretation of mutation types through the fluorescence melting peak signal. In this study, two H. pylori-positive specimens using culture did not show bacterial detection using RT-PCR. We believe that the detection failure was caused by using the leftover tissues on RUT without controlling the incubation times. In fact, neither specimen (each had a bacterial concentration of 8 ng/μL and 5.3 ng/μL) satisfied the cutoff value of appropriate bacterial concentration for the RT-PCR test. In previous studies, when the leftover tissue following RUT was used within 4 hours after inoculation, the H. pylori could be cultured more successfully.14 It is expected that if the RT-PCR assay were performed using fresh gastric tissue, the detection rate might increase. We need further randomized controlled trials with large numbers of patients for further investigations.

CLR resistance affects the eradication rate and is considered the main cause of treatment failure. In previous studies, most point mutations causing CLR resistance by preventing the macrolide from binding were located in A2143G (69.8%), A2142G (11.7%), and A2142C (2.6%).15 Additionally, other mutations such as A2115G, A2142T, G2141A, and T2182C can also influence CLR resistance.15,16 The mutation distributions vary in different regions. In the United States of America, the positivity rates of A2142G and A2143G mutations are 48% to 53% and 39% to 45%, respectively, whereas the A2142C mutation is 0% to 7%. Similarly, in Europe, the A2142G and A2143G mutations are 23% to 33% and 44% to 67%, respectively, and A2142C mutation has been reported as 2% to 10%.17,18 On the other hand, in Japan and China, over 90% and 100%, respectively, of CLR resistance cases were found to have A2143G mutations, although the number of patients was relatively small.19,20 Additionally, most CLR resistance cases in South Korea were confirmed to have the A2143G mutation.21,22

Currently, CLR resistance can detect A2142G and A2143G using this RT-PCR kit. In our study, two specimens that were identified as having CLR resistance in antimicrobial susceptibility tests showed neither resistance nor A2142G or A2143G mutations in RT-PCR. One of these was a quality control failed specimen (concentration of 6.7 ng/μL), whereas the other had suitable quality and quantity for performing RT-PCR. We speculated that the quality and quantity of the samples leftover from RUT might be not suitable for performing RT-PCR. It is also possible that the specimen may have had mutations other than A2142G and A2143G, causing CLR resistance in the antimicrobial susceptibility test. Further analysis is needed to verify the presence of other, abovementioned mutations and to investigate the mutation types that affect clinically significant CLR resistance.

There are several limitations to this study. First, the sample size was relatively small for analyzing the RT-PCR predictive values and accuracy. Second, there was no comparative group to demonstrate that using leftover tissue of RUT as fresh tissue was insufficient for conducting RT-PCR. However, our study did establish that RT-PCR is more appropriate than RUT and agrees well with the antimicrobial susceptibility culturing test.

In conclusion, the RT-PCR assay may be an alternate method for RUT to detect H. pylori infection and can simultaneously and effectively verify the presence of CLR resistance. It is expected that the H. pylori eradication rate will increase if clinicians select treatment regimens based on these results.

This work was supported by a grant from SK Chemical Research Fund of the Korean Society of Gastroenterology in 2020 and the Korean College of Helicobacter and Upper Gastrointestinal Research Foundation (grant number: KCHUGR-202002501).

J.Y.A. is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.

Study concept and design: J.Y.A., J.C. Data acquisition: J.H.N., J.Y.A., J.C., Y.S.P., H.K.N., J.H.L., K.W.J., D.H.K., K.D.C., H.J.S., G.H.L., H.Y.J., J.M.K. Data analysis and interpretation of the data: J.H.N., J.Y.A., J.C. Drafting of the manuscripts: J.H.N., J.Y.A., J.C. Critical revision of the article for intellectual content: J.H.N., J.Y.A., J.C. Obtained funding: J.Y.A. Approval of final manuscript: all authors.

  1. Fock KM, Katelaris P, Sugano K, et al. Second Asia-Pacific Consensus Guidelines for Helicobacter pylori infection. J Gastroenterol Hepatol 2009;24:1587-1600.
    Pubmed CrossRef
  2. Kim SG, Jung HK, Lee HL, et al. Guidelines for the diagnosis and treatment of Helicobacter pylori infection in Korea, 2013 revised edition. J Gastroenterol Hepatol 2014;29:1371-1386.
    Pubmed CrossRef
  3. Malfertheiner P, Megraud F, O'Morain CA, et al. Management of Helicobacter pylori infection: the Maastricht IV/Florence Consensus Report. Gut 2012;61:646-664.
    Pubmed CrossRef
  4. Chung JW, Lee GH, Han JH, et al. The trends of one-week first-line and second-line eradication therapy for Helicobacter pylori infection in Korea. Hepatogastroenterology 2011;58:246-250.
    Pubmed
  5. Yakoob J, Jafri W, Abid S, et al. Role of rapid urease test and histopathology in the diagnosis of Helicobacter pylori infection in a developing country. BMC Gastroenterol 2005;5:38.
    Pubmed KoreaMed CrossRef
  6. Mégraud F, Lehours P. Helicobacter pylori detection and antimicrobial susceptibility testing. Clin Microbiol Rev 2007;20:280-322.
    Pubmed KoreaMed CrossRef
  7. Cerqueira L, Fernandes RM, Ferreira RM, et al. Validation of a fluorescence in situ hybridization method using peptide nucleic acid probes for detection of Helicobacter pylori clarithromycin resistance in gastric biopsy specimens. J Clin Microbiol 2013;51:1887-1893.
    Pubmed KoreaMed CrossRef
  8. Chung JW, Lee GH, Jeong JY, et al. Resistance of Helicobacter pylori strains to antibiotics in Korea with a focus on fluoroquinolone resistance. J Gastroenterol Hepatol 2012;27:493-497.
    Pubmed CrossRef
  9. Lee JH, Ahn JY, Choi KD, et al. Nationwide antibiotic resistance mapping of Helicobacter pylori in Korea: a prospective multicenter study. Helicobacter 2019;24:e12592.
    Pubmed CrossRef
  10. Vaira D, Gatta L, Ricci C, Miglioli M. Review article: diagnosis of Helicobacter pylori infection. Aliment Pharmacol Ther 2002;16 Suppl 1:16-23.
    Pubmed CrossRef
  11. Loy CT, Irwig LM, Katelaris PH, Talley NJ. Do commercial serological kits for Helicobacter pylori infection differ in accuracy? A meta-analysis. Am J Gastroenterol 1996;91:1138-1144.
    Pubmed
  12. Jung DH, Kim JH, Jeong SJ, et al. Peptide nucleic acid probe-based analysis as a new detection method for clarithromycin resistance in Helicobacter pylori. Gut Liver 2018;12:641-647.
    Pubmed KoreaMed CrossRef
  13. Nahm JH, Kim WK, Kwon Y, Kim H. Detection of Helicobacter pylori with clarithromycin resistance-associated mutations using peptide nucleic acid probe-based melting point analysis. Helicobacter 2019;24:e12634.
    Pubmed CrossRef
  14. Gong EJ, Ahn JY, Jung DK, et al. Isolation of Helicobacter pylori using leftover tissue in the rapid urease test kit. Helicobacter 2020;25:e12733.
    Pubmed CrossRef
  15. Mégraud F. H pylori antibiotic resistance: prevalence, importance, and advances in testing. Gut 2004;53:1374-1384.
    Pubmed KoreaMed CrossRef
  16. Hultén K, Gibreel A, Sköld O, Engstrand L. Macrolide resistance in Helicobacter pylori: mechanism and stability in strains from clarithromycin-treated patients. Antimicrob Agents Chemother 1997;41:2550-2553.
    Pubmed KoreaMed CrossRef
  17. Alarcón T, Domingo D, Prieto N, López-Brea M. Clarithromycin resistance stability in Helicobacter pylori: influence of the MIC and type of mutation in the 23S rRNA. J Antimicrob Chemother 2000;46:613-616.
    Pubmed CrossRef
  18. van Doorn LJ, Glupczynski Y, Kusters JG, et al. Accurate prediction of macrolide resistance in Helicobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: multicenter validation study. Antimicrob Agents Chemother 2001;45:1500-1504.
    Pubmed KoreaMed CrossRef
  19. Kato S, Fujimura S, Udagawa H, et al. Antibiotic resistance of Helicobacter pylori strains in Japanese children. J Clin Microbiol 2002;40:649-653.
    Pubmed KoreaMed CrossRef
  20. Pan ZJ, Su WW, Tytgat GN, Dankert J, van der Ende A. Assessment of clarithromycin-resistant Helicobacter pylori among patients in Shanghai and Guangzhou, China, by primer-mismatch PCR. J Clin Microbiol 2002;40:259-261.
    Pubmed KoreaMed CrossRef
  21. An B, Moon BS, Kim H, et al. Antibiotic resistance in Helicobacter pylori strains and its effect on H. pylori eradication rates in a single center in Korea. Ann Lab Med 2013;33:415-419.
    Pubmed KoreaMed CrossRef
  22. Chung WC, Jung SH, Oh JH, et al. Dual-priming oligonucleotide-based multiplex PCR using tissue samples in rapid urease test in the detection of Helicobacter pylori infection. World J Gastroenterol 2014;20:6547-6553.
    Pubmed KoreaMed CrossRef

Article

ahead

Gut and Liver

Published online July 20, 2022

Copyright © Gut and Liver.

Real-Time Polymerase Chain Reaction for the Detection of Helicobacter pylori and Clarithromycin Resistance

Jin Hee Noh1 , Ji Yong Ahn1 , Jene Choi2 , Young Soo Park2 , Hee Kyong Na1 , Jeong Hoon Lee1 , Kee Wook Jung1 , Do Hoon Kim1 , Kee Don Choi1 , Ho June Song1 , Gin Hyug Lee1 , Hwoon-Yong Jung1 , Jung Mogg Kim3

Departments of 1Gastroenterology and 2Pathology, Asan Medical Center, University of Ulsan College of Medicine, and 3Department of Microbiology, Hanyang University College of Medicine, Seoul, Korea

Correspondence to:Ji Yong Ahn
ORCID https://orcid.org/0000-0002-0030-3744
E-mail ji110@hanmail.net

Received: February 24, 2022; Revised: March 30, 2022; Accepted: April 14, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background/Aims: Real-time polymerase chain reaction (RT-PCR) is a fast and simple method for the simultaneous detection of clarithromycin (CLR) resistance and Helicobacter pylori. We evaluated the effectiveness of RT-PCR compared to that of the rapid urease test (RUT) and assessed its value in verifying CLR resistance.
Methods: A total of 70 specimens with confirmed H. pylori infection in culture were enrolled and analyzed in this prospective study. All specimens were subjected to RT-PCR assay using fluorescence melting peak signals to detect H. pylori infection and CLR resistances caused by either A2142G or A2143G mutations in the 23S ribosomal RNA gene (23S rRNA). The results were compared to those of RUT and antimicrobial susceptibility culturing tests to investigate the efficacy of RT-PCR.
Results: Among the 70 specimens analyzed, the positivity rate was 97.1% (68/70) with RT-PCR and 82.9% (58/70) with RUT. CLR resistance (minimum inhibitory concentration >1.0 μg/mL) was confirmed in 18.6% (13/70), and fluorescence melting curve analysis showed that 84.6% (11/13) had point mutations in 23S rRNA. Ten specimens had only A2143G mutation, and one specimen contained both A2142G and A2143G mutations.
Conclusions: RT-PCR assay was found to be more efficient than RUT in detecting H. pylori infection and could effectively verify CLR resistance compared to the antimicrobial susceptibility culturing test. Considering the high sensitivity and accessibility of RT-PCR method, it could be used to easily detect CLR-resistant H. pylori, thus helping clinicians select suitable treatment regimen and improve the eradication rate.

Keywords: Clarithromycin resistance, Helicobacter pylori, Culture, Rapid urease test, Real-time polymerase chain reaction

INTRODUCTION

Helicobacter pylori is a Gram-negative, spiral-shaped bacterium that colonizes the gastric mucosa and may cause atrophic gastritis, peptic ulcers, gastric cancer, and mucosal-associated lymphoid tissue lymphoma. Therefore, eradication of H. pylori is important not only for patients infected with the disease but also for asymptomatic, healthy individuals. With a global increase in antibiotic resistance, the success rate of H. pylori eradication is decreasing.1-3 The main cause of treatment failure has been reported as clarithromycin (CLR) resistance, and among Korean patients, CLR resistance has gradually increased from 13.7% in 2003 to 16.7% in 2006 and 23.7% in 2012.4 Thus, it is important to detect CLR resistance and select an appropriate treatment regimen to improve the H. pylori eradication rate.

The rapid urease test (RUT) is the most common method for detecting H. pylori infection. It yields results quickly, has high sensitivity and specificity, and is economic and simple to use. However, it requires a high density of bacteria and cannot detect antibiotic resistance.5 Culture and antibiotic susceptibility tests can confirm antibiotic resistance regardless of genetic mutations, but culturing bacterial takes at least 5 to 7 days. Additionally, because the culture conditions and composition environments may vary depending on the institution and are difficult to construct, the culture success rate ranges from 50% to 80%.6 However, the real-time polymerase chain reaction (RT-PCR) assay is a fast and simple method for the simultaneous detection of CLR resistance and H. pylori.7 Although it is a sensitive and specific method for detecting H. pylori infection in gastric tissue samples, there is a lack of studies comparing the RT-PCR assay, RUT, and antibiotic susceptibility test using conventional culture.

Therefore, we evaluated whether the RT-PCR assay is more efficient than RUT for detecting H. pylori and whether it is appropriate for identifying CLR resistance with the associated mutations.

MATERIALS AND METHODS

1. Study design and population

This prospective study was conducted between December 2019 and December 2020 at Asan Medical Center, Seoul, Korea. This study was approved by the Institutional Review Board of Asan Medical Center (IRB number: 2020-0082) and conducted in accordance with the standards of the Declaration of Helsinki. The patients, who were between 19 and 80 years old and had no history of H. pylori eradication, were enrolled after providing written informed consent. Patients with a history of gastric surgery, hematologic disease with bleeding tendency, or antibiotics administration within the past month were excluded.

All patients underwent serum H. pylori immunoglobulin G (IgG) testing and esophagogastroduodenoscopy with pairs of mucosal biopsies from gastric antrum and corpus for each RUT and culture with antimicrobial susceptibility tests. Of these, the specimens from patients who were positive for either antrum or body on RUT were used for the RT-PCR assay with U-TOPTM HPy & ClaR Detection Kit (SeaSun Biomaterials, Daejeon, Korea). Finally, the 70 specimens that were identified as having H. pylori infection in culture, which was considered a confident method, were analyzed to investigate the H. pylori detection of RT-PCR assay and RUT, as well as the presence of CLR resistances by detecting A2142G or A2143G mutations in the 23S ribosomal RNA gene (23S rRNA) (Supplementary Fig. 1).

2. H. pylori culture

The tissues obtained from gastric antrum and corpus during esophagogastroduodenoscopy were first placed in a sterile Eppendorf tube and then placed in a vacuum bottle containing dry ice. The Eppendorf tubes were stored in a –80°C deep freezer and the tissues were allowed to thaw at room temperature before analysis. The specimens were inoculated onto an H. pylori isolation medium (Brucella broth agar supplemented with 5% sheep blood and containing 10 μg/L vancomycin, 5 μg/L trimethoprim, 5 μg/L amphotericin B, and 2.5 IU polymyxin B) and incubated at 37°C under microaerophilic conditions (5% O2, 10% CO2, and 85% N2) for 5 to 7 days. Suspected H. pylori colonies were identified based on morphology, Gram staining, and urease positive reaction.

The minimum inhibitory concentrations (MICs) of antibiotics were determined using the serial 2-fold agar dilution method as described previously.8 Bacteria were sub-cultured for 48 hours on Mueller–Hinton agar supplemented with 5% defibrinated sheep blood. The bacterial suspension was adjusted to 1×107 colony-forming units and inoculated to each antibiotic-containing agar dilution plate followed by incubation for 3 days at 37°C under microaerophilic conditions. The MIC of antibiotics was evaluated after 72 hours. A standard H. pylori strain (ATCC 43504) was used as the control. Resistance breakpoint for CLR was set as >1 μg/mL.9

3. RT-PCR for detection of H. pylori infection and CLR resistance

According to the manufacturer's instruction (QIAamp DNA Tissue Kit; Qiagen, Hilden, Germany), the genomic DNA was extracted from the leftover biopsied samples after performing the RUT. The quality and quantity of the genomic DNA samples were evaluated using a Nanodrop spectrophotometers (Thermo Fisher, Waltham, MA, USA), with a >15 ng/μL cutoff value of appropriate bacterial concentration for the test. RT-PCR for detection of H. pylori and CLR resistances was performed with the U-TOPTM HPy & ClaR Detection Kit (SeaSun Biomaterials) using the CFX96 Real-Time PCR detection system V1.6 (Bio-Rad, Hercules, CA, USA). Specific primers were used to amplify the H. pylori genes. The polymerase nucleic acid probe-based qPCR method was used for analyzing the mutations status of H. pylori. Data were analyzed using the SS AnalyzerII V1.0 (SeaSun Biomaterials, Seoul, Korea) software. Infection and CLR resistance were differentiated by the fluorescence melting peak signal (Tm) (Fig. 1A). For cases with mixed infection of wild type and mutant H. pylori, melt curve analysis was further performed to distinguish two different peaks according to each percentage, and the presence of point mutation was differentiated using the gradient difference (Fig. 1B and C).

Figure 1. Representative results of internal positive control (IPC), Helicobacter pylori-positive, A2142G mutation, and A2143G mutation using U-TOPTM HPy & ClaR Detection Kit. (A) Examples of melting peak signal (Tm) with standard value. (B) Tm results according to the percentage of wild type and mutant (A2142G and A2143G point mutations) in mixed-infection samples, and (C) examples with different percentage.

4. Statistical analysis

Descriptive statistics for the categorical variables were summarized as proportions, and the continuous variables were summarized using the medians (interquartile range). The McNemar test was applied to calculate the diagnostic sensitivity of each test. The sensitivity (%) was calculated as follows: 100×true positives/(true positives+false negatives). The true positives and false negatives were determined depending on whether the H. pylori infection was confirmed by culture. The specificity (%) of the method was calculated as follows: 100×true negatives/(false positives+true negatives), where true negatives and false positives were defined according to H. pylori infection determined by combining the results of culture and H. pylori IgG antibody test. All statistical analyses were conducted using SPSS version 24 (IBM Corp., Armonk, NY, USA).

RESULTS

1. Clinical characteristics and diagnostic tests

A total of 70 specimens from 39 patients were included in this study. The median age was 62 years (interquartile range, 58 to 70 years), and 87% of the patients (34/39) were male. Indications of test were 56% (22/39) of dysplasia and 44% (17/39) of early gastric cancer. The RUT results showed 72% (23/32) antrum positivity and 92% (35/38) body positivity, whereas the RT-PCR results showed 94% (30/32) antrum positivity and 100% (38/38) body positivity. The results of H. pylori IgG antibody test, RUT, and RT-PCR of the 70 samples are summarized in Supplementary Table 1.

2. Comparison of H. pylori detection rate using RUT and RT-PCR

Among the 70 specimens, RT-PCR and RUT assays showed sensitivities of 97.1% (68/70) and 82.9% (58/70) (Fig. 2), and specificities of 100% (8/8) and 62.5% (5/8), respectively. Table 1 shows the accordance of each H. pylori diagnostic test according to the stomach location, namely, the antrum and body. The RUT and RT-PCR assay showed accordance of 70% (21/30) for the antrum and 92% (35/38) for the body. The RUT and culture represented 72% (23/32) and 92% (35/38) of accordance for the antrum and body, respectively. In the case of RT-PCR and culture, the accordance was 94% (30/32) for the antrum and 100% (38/38) for the body. Consequently, the body specimens showed higher accordance between the two tests than the antrum specimens.

Table 1 . Accordance among RUT, RT-PCR, and Culture for Helicobacter pylori Diagnosis According to Its Location in the Stomach of Culture-Positive Specimens (n=70).

VariableRUTRT-PCR
AntrumBodyAntrumBody
Culture (n=70)Antrum (n=32)23 (72)30 (94)
Body (n=38)35 (92)38 (100)
RT-PCR (n=68)Antrum (n=30)21 (70)**
Body (n=38)35 (92)**

Data are presented as number (%)..

RUT, rapid urease test; RT-PCR, real-time polymerase chain reaction..

*Comparisons of the same test (RT-PCR)..


Figure 2. Comparison of Helicobacter pylori detection rates between the rapid urease test (RUT) and real-time polymerase chain reaction (RT-PCR) assay in culture-positive specimens.

3. CLR resistance by culture and RT-PCR

Fig. 3 shows the CLR resistance in culture and the proportions of point mutations. Among the 70 specimens, 13 (18.6%) were CLR resistant (MIC >1.0 μg/mL) and 57 (82.3%) were CLR susceptible. Of the CLR-resistant specimens, 11 (84.6%) had 23S ribosomal RNA point mutations, detected using fluorescence melting curve analysis. The A2143G mutation was observed in 10 specimens, and notably, one specimen showed A2142G and A2143G double mutations. The MIC of CLR and the mutation types of each specimen are summarized in Table 2.

Table 2 . MIC of CLR and Mutation Types Detected Using RT-PCR Assay on CLR-Resistant Specimens.

Specimen numberCLR MIC, mg/LMutation type
1>128A2142G, A2143G
2>128A2143G
364A2143G
464A2143G
532A2143G
616A2143G
716A2143G
816A2143G
916A2143G
1016A2143G
118A2143G
128-
138-

MIC, minimum inhibitory concentration; CLR, clarithromycin; RT-PCR, real-time polymerase chain reaction..

RT-PCR was performed using the U-TOPTM HPy & ClaR Detection Kit (SeaSun Biomaterials)..


Figure 3. Clarithromycin (CLR) resistance and proportion of point mutations (A2142G and A2143G) confirmed using agar dilution test and real-time polymerase chain reaction (RT-PCR).

DISCUSSION

Although several PCR-based H. pylori detection methods with high sensitivity and specificity have recently been reported, few studies have clinically focused on the comparison between PCR and other methods such as RUT and standard susceptibility culturing tests. In this study, we investigated whether the RT-PCR assay is more efficient than the RUT for detecting H. pylori and whether it is more appropriate for identifying CLR resistance compared to the susceptibility culturing test. Among the 70 specimens with confirmed H. pylori infection in culture, 97.1% and 82.9% were positively confirmed using RT-PCR and RUT, respectively. The CLR resistance in culture was confirmed in 18.6% of the specimens, and 84.6% of them had A2142G and A2143G mutations in the 23S rRNA gene, as detected using the fluorescence melting curve analysis. Additionally, H. pylori IgG antibody test was performed using all specimens, of which, four were found to be negative. The sensitivity of H. pylori IgG antibody test was 94.3% (66/70), similar to those reported in previous studies.10,11

According to previous studies, the results of H. pylori detection and CLR resistance of RT-PCR-based assays were similar to those of the commercialized DPO-PCR (Seeplex ClaR-H. pylori ACE Detection; Seegene Institute of Life Sciences, Seoul, Korea) and conventional PCR.12,13 RT-PCR-based assay is a simple and accurate method for detecting H. pylori and mutation types of CLR resistance that can be analyzed using a small formalin-fixed, paraffin-embedded specimen after amplification. As it is a one-step analysis, it can avoid cross-contamination that can occur during electrophoresis. Moreover, RT-PCR allows easy and accurate interpretation of mutation types through the fluorescence melting peak signal. In this study, two H. pylori-positive specimens using culture did not show bacterial detection using RT-PCR. We believe that the detection failure was caused by using the leftover tissues on RUT without controlling the incubation times. In fact, neither specimen (each had a bacterial concentration of 8 ng/μL and 5.3 ng/μL) satisfied the cutoff value of appropriate bacterial concentration for the RT-PCR test. In previous studies, when the leftover tissue following RUT was used within 4 hours after inoculation, the H. pylori could be cultured more successfully.14 It is expected that if the RT-PCR assay were performed using fresh gastric tissue, the detection rate might increase. We need further randomized controlled trials with large numbers of patients for further investigations.

CLR resistance affects the eradication rate and is considered the main cause of treatment failure. In previous studies, most point mutations causing CLR resistance by preventing the macrolide from binding were located in A2143G (69.8%), A2142G (11.7%), and A2142C (2.6%).15 Additionally, other mutations such as A2115G, A2142T, G2141A, and T2182C can also influence CLR resistance.15,16 The mutation distributions vary in different regions. In the United States of America, the positivity rates of A2142G and A2143G mutations are 48% to 53% and 39% to 45%, respectively, whereas the A2142C mutation is 0% to 7%. Similarly, in Europe, the A2142G and A2143G mutations are 23% to 33% and 44% to 67%, respectively, and A2142C mutation has been reported as 2% to 10%.17,18 On the other hand, in Japan and China, over 90% and 100%, respectively, of CLR resistance cases were found to have A2143G mutations, although the number of patients was relatively small.19,20 Additionally, most CLR resistance cases in South Korea were confirmed to have the A2143G mutation.21,22

Currently, CLR resistance can detect A2142G and A2143G using this RT-PCR kit. In our study, two specimens that were identified as having CLR resistance in antimicrobial susceptibility tests showed neither resistance nor A2142G or A2143G mutations in RT-PCR. One of these was a quality control failed specimen (concentration of 6.7 ng/μL), whereas the other had suitable quality and quantity for performing RT-PCR. We speculated that the quality and quantity of the samples leftover from RUT might be not suitable for performing RT-PCR. It is also possible that the specimen may have had mutations other than A2142G and A2143G, causing CLR resistance in the antimicrobial susceptibility test. Further analysis is needed to verify the presence of other, abovementioned mutations and to investigate the mutation types that affect clinically significant CLR resistance.

There are several limitations to this study. First, the sample size was relatively small for analyzing the RT-PCR predictive values and accuracy. Second, there was no comparative group to demonstrate that using leftover tissue of RUT as fresh tissue was insufficient for conducting RT-PCR. However, our study did establish that RT-PCR is more appropriate than RUT and agrees well with the antimicrobial susceptibility culturing test.

In conclusion, the RT-PCR assay may be an alternate method for RUT to detect H. pylori infection and can simultaneously and effectively verify the presence of CLR resistance. It is expected that the H. pylori eradication rate will increase if clinicians select treatment regimens based on these results.

SUPPLEMENTARY MATERIALS

Supplementary materials can be accessed at https://doi.org/10.5009/gnl220076.

Supplementary material.pdf

ACKNOWLEDGEMENTS

This work was supported by a grant from SK Chemical Research Fund of the Korean Society of Gastroenterology in 2020 and the Korean College of Helicobacter and Upper Gastrointestinal Research Foundation (grant number: KCHUGR-202002501).

CONFLICTS OF INTEREST

J.Y.A. is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.

AUTHOR CONTRIBUTIONS

Study concept and design: J.Y.A., J.C. Data acquisition: J.H.N., J.Y.A., J.C., Y.S.P., H.K.N., J.H.L., K.W.J., D.H.K., K.D.C., H.J.S., G.H.L., H.Y.J., J.M.K. Data analysis and interpretation of the data: J.H.N., J.Y.A., J.C. Drafting of the manuscripts: J.H.N., J.Y.A., J.C. Critical revision of the article for intellectual content: J.H.N., J.Y.A., J.C. Obtained funding: J.Y.A. Approval of final manuscript: all authors.

Fig 1.

Figure 1.Representative results of internal positive control (IPC), Helicobacter pylori-positive, A2142G mutation, and A2143G mutation using U-TOPTM HPy & ClaR Detection Kit. (A) Examples of melting peak signal (Tm) with standard value. (B) Tm results according to the percentage of wild type and mutant (A2142G and A2143G point mutations) in mixed-infection samples, and (C) examples with different percentage.
Gut and Liver 2022; :

Fig 2.

Figure 2.Comparison of Helicobacter pylori detection rates between the rapid urease test (RUT) and real-time polymerase chain reaction (RT-PCR) assay in culture-positive specimens.
Gut and Liver 2022; :

Fig 3.

Figure 3.Clarithromycin (CLR) resistance and proportion of point mutations (A2142G and A2143G) confirmed using agar dilution test and real-time polymerase chain reaction (RT-PCR).
Gut and Liver 2022; :

Table 1 Accordance among RUT, RT-PCR, and Culture for Helicobacter pylori Diagnosis According to Its Location in the Stomach of Culture-Positive Specimens (n=70)

VariableRUTRT-PCR
AntrumBodyAntrumBody
Culture (n=70)Antrum (n=32)23 (72)30 (94)
Body (n=38)35 (92)38 (100)
RT-PCR (n=68)Antrum (n=30)21 (70)**
Body (n=38)35 (92)**

Data are presented as number (%).

RUT, rapid urease test; RT-PCR, real-time polymerase chain reaction.

*Comparisons of the same test (RT-PCR).


Table 2 MIC of CLR and Mutation Types Detected Using RT-PCR Assay on CLR-Resistant Specimens

Specimen numberCLR MIC, mg/LMutation type
1>128A2142G, A2143G
2>128A2143G
364A2143G
464A2143G
532A2143G
616A2143G
716A2143G
816A2143G
916A2143G
1016A2143G
118A2143G
128-
138-

MIC, minimum inhibitory concentration; CLR, clarithromycin; RT-PCR, real-time polymerase chain reaction.

RT-PCR was performed using the U-TOPTM HPy & ClaR Detection Kit (SeaSun Biomaterials).


References

  1. Fock KM, Katelaris P, Sugano K, et al. Second Asia-Pacific Consensus Guidelines for Helicobacter pylori infection. J Gastroenterol Hepatol 2009;24:1587-1600.
    Pubmed CrossRef
  2. Kim SG, Jung HK, Lee HL, et al. Guidelines for the diagnosis and treatment of Helicobacter pylori infection in Korea, 2013 revised edition. J Gastroenterol Hepatol 2014;29:1371-1386.
    Pubmed CrossRef
  3. Malfertheiner P, Megraud F, O'Morain CA, et al. Management of Helicobacter pylori infection: the Maastricht IV/Florence Consensus Report. Gut 2012;61:646-664.
    Pubmed CrossRef
  4. Chung JW, Lee GH, Han JH, et al. The trends of one-week first-line and second-line eradication therapy for Helicobacter pylori infection in Korea. Hepatogastroenterology 2011;58:246-250.
    Pubmed
  5. Yakoob J, Jafri W, Abid S, et al. Role of rapid urease test and histopathology in the diagnosis of Helicobacter pylori infection in a developing country. BMC Gastroenterol 2005;5:38.
    Pubmed KoreaMed CrossRef
  6. Mégraud F, Lehours P. Helicobacter pylori detection and antimicrobial susceptibility testing. Clin Microbiol Rev 2007;20:280-322.
    Pubmed KoreaMed CrossRef
  7. Cerqueira L, Fernandes RM, Ferreira RM, et al. Validation of a fluorescence in situ hybridization method using peptide nucleic acid probes for detection of Helicobacter pylori clarithromycin resistance in gastric biopsy specimens. J Clin Microbiol 2013;51:1887-1893.
    Pubmed KoreaMed CrossRef
  8. Chung JW, Lee GH, Jeong JY, et al. Resistance of Helicobacter pylori strains to antibiotics in Korea with a focus on fluoroquinolone resistance. J Gastroenterol Hepatol 2012;27:493-497.
    Pubmed CrossRef
  9. Lee JH, Ahn JY, Choi KD, et al. Nationwide antibiotic resistance mapping of Helicobacter pylori in Korea: a prospective multicenter study. Helicobacter 2019;24:e12592.
    Pubmed CrossRef
  10. Vaira D, Gatta L, Ricci C, Miglioli M. Review article: diagnosis of Helicobacter pylori infection. Aliment Pharmacol Ther 2002;16 Suppl 1:16-23.
    Pubmed CrossRef
  11. Loy CT, Irwig LM, Katelaris PH, Talley NJ. Do commercial serological kits for Helicobacter pylori infection differ in accuracy? A meta-analysis. Am J Gastroenterol 1996;91:1138-1144.
    Pubmed
  12. Jung DH, Kim JH, Jeong SJ, et al. Peptide nucleic acid probe-based analysis as a new detection method for clarithromycin resistance in Helicobacter pylori. Gut Liver 2018;12:641-647.
    Pubmed KoreaMed CrossRef
  13. Nahm JH, Kim WK, Kwon Y, Kim H. Detection of Helicobacter pylori with clarithromycin resistance-associated mutations using peptide nucleic acid probe-based melting point analysis. Helicobacter 2019;24:e12634.
    Pubmed CrossRef
  14. Gong EJ, Ahn JY, Jung DK, et al. Isolation of Helicobacter pylori using leftover tissue in the rapid urease test kit. Helicobacter 2020;25:e12733.
    Pubmed CrossRef
  15. Mégraud F. H pylori antibiotic resistance: prevalence, importance, and advances in testing. Gut 2004;53:1374-1384.
    Pubmed KoreaMed CrossRef
  16. Hultén K, Gibreel A, Sköld O, Engstrand L. Macrolide resistance in Helicobacter pylori: mechanism and stability in strains from clarithromycin-treated patients. Antimicrob Agents Chemother 1997;41:2550-2553.
    Pubmed KoreaMed CrossRef
  17. Alarcón T, Domingo D, Prieto N, López-Brea M. Clarithromycin resistance stability in Helicobacter pylori: influence of the MIC and type of mutation in the 23S rRNA. J Antimicrob Chemother 2000;46:613-616.
    Pubmed CrossRef
  18. van Doorn LJ, Glupczynski Y, Kusters JG, et al. Accurate prediction of macrolide resistance in Helicobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: multicenter validation study. Antimicrob Agents Chemother 2001;45:1500-1504.
    Pubmed KoreaMed CrossRef
  19. Kato S, Fujimura S, Udagawa H, et al. Antibiotic resistance of Helicobacter pylori strains in Japanese children. J Clin Microbiol 2002;40:649-653.
    Pubmed KoreaMed CrossRef
  20. Pan ZJ, Su WW, Tytgat GN, Dankert J, van der Ende A. Assessment of clarithromycin-resistant Helicobacter pylori among patients in Shanghai and Guangzhou, China, by primer-mismatch PCR. J Clin Microbiol 2002;40:259-261.
    Pubmed KoreaMed CrossRef
  21. An B, Moon BS, Kim H, et al. Antibiotic resistance in Helicobacter pylori strains and its effect on H. pylori eradication rates in a single center in Korea. Ann Lab Med 2013;33:415-419.
    Pubmed KoreaMed CrossRef
  22. Chung WC, Jung SH, Oh JH, et al. Dual-priming oligonucleotide-based multiplex PCR using tissue samples in rapid urease test in the detection of Helicobacter pylori infection. World J Gastroenterol 2014;20:6547-6553.
    Pubmed KoreaMed CrossRef
Gut and Liver

Vol.16 No.5
September, 2022

pISSN 1976-2283
eISSN 2005-1212

qrcode
qrcode

Supplementary

Share this article on :

  • line

Popular Keywords

Gut and LiverQR code Download
qr-code

Editorial Office