Skip to main content

Detection of four clarithromycin resistance point mutations in Helicobacter pylori: comparison of real-time PCR and PCR-RFLP methods

Abstract

Helicobacter pylori infection can be eradicated in up to 90 % of patients using current combination of triple therapies, of which clarithromycin is a key component. The development of clarithromycin resistance in H. pylori is recognized as a significant contributing factor in treatment failure. The aim of this study is to compare two fast and direct diagnostic methods to evaluate clarithromycin resistance in gastric biopsy specimens. This cross-sectional descriptive study was performed on 150 gastric biopsy specimens. Analysis for clarithromycin resistance was performed by real-time polymerase chain reaction (PCR) assay for A2144G, A2143G, A2143C, and A2142G point mutations by specific probes. Also, A2142G and A2143G point mutations were detected by PCR-RFLP method and using BsaI and MboII restriction enzymes. Out of 150 samples, 96 (64 %) clarithromycin-sensitive- and 54 (36 %) clarithromycin-resistant strains were detected by TaqMan real-time PCR assay. Thirty-seven (24.67 %) resistant strains were detected by PCR-RFLP method. Results showed that real-time PCR assay has enough accuracy to identify clarithromycin resistance and their mutations in a short time.

This is a preview of subscription content, access via your institution.

Fig. 1

References

  • Alfaresi MS, Abdulsalam AI, Elkoush AA (2007) Analysis of Helicobacter pylori antimicrobial susceptibility and virulence genes in gastric mucosal biopsies in the United Arab Emirates. Indian J Gastroenterol 26:221–224

    PubMed  Google Scholar 

  • Ben Mansour K, Burucoa C, Zribi M, Masmoudi A, Karoui S, Kallel L, Chouaib S, Matri S, Fekih M, Zarrouk S, Labbene M, Boubaker J, Cheikh I, Hriz MB, Siala N, Ayadi A, Filali A, Mami NB, Najjar T, Maherzi A, Sfar MT, Fendri C (2010) Primary resistance to clarithromycin, metronidazole and amoxicillin of Helicobacter pylori isolated from Tunisian patients with peptic ulcers and gastritis: a prospective multicentre study. Ann Clin Microbiol and Antimicrob 9:22. Available online at: http://www.ann-clinmicrob.com/content/9/1/22

    Google Scholar 

  • Burucoa C, Garnier M, Silvain C, Fauchere JL (2008) Quadruplex real-time PCR assay using allele-specific scorpion primers for detection of mutations conferring clarithromycin resistance to Helicobacter pylori. J Clin Microbiol 46:2320–2326

    PubMed  Article  CAS  Google Scholar 

  • Chisholm SA, Owen RJ, Teare EL, Saverymuttu S (2001) PCR-based diagnosis of Helicobacter pylori infection and real-time determination of clarithromycin resistance directly from human gastric biopsy samples. J Clin Microbiol 39:1217–1220

    PubMed  Article  CAS  Google Scholar 

  • Cirak MY, Akyön Y, Mégraud F (2007) Diagnosis of Helicobacter pylori. Helicobacter 12:4–9

    PubMed  Article  CAS  Google Scholar 

  • De Francesco V, Margiotta M, Zullo A, Hassan C, Valle ND, Burattini O, Cea U, Stoppino G, Amoruso A, Stella F, Morini S, Panella C, Ierardi E (2006) Primary clarithromycin resistance in Italy assessed on Helicobacter pylori DNA sequences by TaqMan real-time polymerase chain reaction. Aliment Pharmacol Ther 23:429–435

    PubMed  Article  Google Scholar 

  • De Francesco V, Zullo A, Ierardi E, Giorgio F, Perna F, Hassan C, Morini S, Panella C, Vaira D (2010) Phenotypic and genotypic Helicobacter pylori clarithromycin resistance and therapeutic outcome: benefits and limits. J Antimicrob Chemother 65:327–332

    PubMed  Article  Google Scholar 

  • Demir M, Gokturk HS, Ozturk NA, Arslan H, Serin E, Yilmaz U (2009) Clarithromycin resistance and efficacy of clarithromycin-containing triple eradication therapy for Helicobacter pylori infection in type 2 diabetes mellitus patients. South Med J 102:116–120

    Article  Google Scholar 

  • Elviss NC, Lawson AJ, Owen RJ (2004) Application of 3′-mismatched reverse primer PCR compared with real-time PCR and PCR-RFLP for the rapid detection of 23S rDNA mutations associated with clarithromycin resistance in Helicobacter pylori. Int J Antimicrob Agents 23:349–355

    PubMed  Article  CAS  Google Scholar 

  • Falsafi T, Mobasheri F, Nariman F, Najafi M (2004) Susceptibilities to different antibiotics of Helicobacter pylori strains isolated from patients at the Pediatric Medical Center of Tehran, Iran. J Clin Microbiol 42:387–389

    PubMed  Article  CAS  Google Scholar 

  • Kargar M, Baghernejad M, Doosti A (2010) Role of NADPH-insensitive nitroreductase gene to metronidazole resistance of Helicobacter pylori strains. DARU 18:137–140

    PubMed  CAS  Google Scholar 

  • Kargar M, Souod N, Ghorbani-Dalini S, Doosti A, Rezaeian AA (2011a) Evaluation of cagA tyrosine phosphorylation DNA motifs in Helicobacter pylori isolates from gastric disorder patients in west of Iran. Sci Res Essays 6:6454–6458

    CAS  Google Scholar 

  • Kargar M, Souod N, Doosti A, Ghorbani-Dalini S (2011b) Prevalence of Helicobacter pylori vacuolating cytotoxin A gene as a predictive marker for different gastrodoudenal diseases. Iran J Clin Infect Dis 6:85–89

    Google Scholar 

  • Kargar M, Baghernejad M, Doosti S, Ghorbani-Dalini S (2011c) Clarithromycin resistance and 23S rRNA mutations in Helicobacter pylori isolates in Iran. Afr J Microbiol Res 5:853–856

    CAS  Google Scholar 

  • Kargar M, Ghorbani-Dalini S, Doosti A, Baghernejad M (2011d) Molecular assessment of clarithromycin resistant Helicobacter pylori strains using rapid and accurate PCR-RFLP method in gastric specimens in Iran. Afr J Biotechnol 10:7675–7678

    CAS  Google Scholar 

  • Kargar M, Ghorbani-Dalini S, Doosti A, Souod N (2012) Real-time PCR for Helicobacter pylori quantification and detection of clarithromycin resistance in gastric tissues from patients with gastrointestinal disorders. Res Microbiol 163:109–113

    PubMed  Article  CAS  Google Scholar 

  • Kato S, Fujimura S, Udagawa H, Shimizu T, Maisawa S, Ozawa K, Linuma K (2002) Antibiotic resistance of Helicobacter pylori strains in Japanese children. J Clin Microbiol 40:649–653

    PubMed  Article  CAS  Google Scholar 

  • Lascols C, Lamarque D, Costa JM, Copie-Bergman C, Glaunec JML, Deforges L, Soussy CJ, Petit JC, Delchier JC, Tankovic J (2003) Fast and accurate quantitative detection of Helicobacter pylori and identification of clarithromycin resistance mutations in H. pylori isolates from gastric biopsy specimens by real-time PCR. J Clin Microbiol 41:4573–4577

    PubMed  Article  CAS  Google Scholar 

  • Liu Z, Shen J, Zhang L, Shen L, Li Q, Zhang B, Zhou J, Gu L, Feng G, Ma J, You WC, Deng D (2008) Prevalence of A2143G mutation of H. pylori-23S rRNA in Chinese subjects with and without clarithromycin use history. BMC Microbiol 8:81. doi:10.1186/1471-2180-8-81

    PubMed  Article  Google Scholar 

  • Lottspeich C, Schwarzer A, Panthel K, Koletzko S, Russmann H (2007) Evaluation of the novel Helicobacter pylori ClariRes real-time PCR assay for detection and clarithromycin susceptibility testing of H. pylori in stool specimens from symptomatic children. J Clin Microbiol 45:1718–1722

    PubMed  Article  CAS  Google Scholar 

  • Marais A, Monteiro L, Occhialini A, Pina M, Lamouliatte H, Mégraud F (1999) Direct detection of Helicobacter pylori resistance to macrolides by a polymerase chain reaction/DNA enzyme immunoassay in gastric biopsy specimens. Gut 44:463–467

    PubMed  Article  CAS  Google Scholar 

  • Matsuoka M, Yoshida Y, Hayakawa K, Fukuchi S, Sugano K (1999) Simultaneous colonisation of Helicobacter pylori with and without mutations in the 23S rRNA gene in patients with no history of clarithromycin exposure. Gut 45:503–507

    PubMed  Article  CAS  Google Scholar 

  • Oleastro M, Menard A, Santos A, Lamouliatte H, Monteiro L, Barthelemy P, Mégraud F (2003) Real-time PCR assay for rapid and accurate detection of point mutations conferring resistance to clarithromycin in Helicobacter pylori. J Clin Microbiol 41:397–402

    PubMed  Article  CAS  Google Scholar 

  • Pan ZJ, Su WW, Tytgat GNJ, Dankert J, Van Der Ende AA (2002) Assessment of clarithromycin-resistant Helicobacter pylori among patients in Shanghai and Guangzhou, China, by Primer-Mismatch PCR. J Clin Microbiol 40:259–261

    PubMed  Article  CAS  Google Scholar 

  • Pina M, Occhialini A, Monteiro L, Doermann HP, Megraud F (1998) Detection of point mutations associated with resistance of Helicobacter pylori to clarithromycin by hybridization in liquid phase. J Clin Microbiol 36:3285–3290

    PubMed  CAS  Google Scholar 

  • Razaghi M, Boutorabi SM, Mirjalili A, Norolahi S, Hashemi M, Jalalian M (2010) Diagnosis of Helicobacter pylori infection by ELISA stool antigen and comparison with the other diagnostic methods. Health Med 4:545–551

    Google Scholar 

  • Ribeiro ML, Ecclissato CC, Mattos RG, Mendonca S, Pedrazzoli J Jr (2007) Quantitative real-time PCR for the clinical detection of Helicobacter pylori. Genet Mol Biol 30:431–434

    Article  CAS  Google Scholar 

  • Schabereiter-Gurtner C, Hirschl AM, Dragosics B, Hufnagl P, Puz S, Kovach Z, Rotter M, Makristathis A (2004) Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy specimens. J Clin Microbiol 42:4512–4518

    PubMed  Article  CAS  Google Scholar 

  • Taneike I, Suzuki K, Nakagawa S, Yamamoto T (2004) Intrafamilial spread of the same clarithromycin-resistant Helicobacter pylori infection confirmed by molecular analysis. J Clin Microbiol 42:3901–3903

    PubMed  Article  CAS  Google Scholar 

  • Taylor DE, Ge Z, Purych D, Lo T, Hiratsuka K (1997) Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations. Antimicrob Agents Chemother 41:2621–2628

    PubMed  CAS  Google Scholar 

  • Van Der Ende A, Van Doorn LJ, Rooijakkers S, Feller M, Tytgat GNJ, Dankert J (2001) Clarithromycin-susceptible and -resistant Helicobacter pylori isolates with identical randomly amplified polymorphic DNA-PCR genotypes cultured from single gastric biopsy specimens prior to antibiotic therapy. J Clin Microbiol 39:2648–2651

    PubMed  Article  Google Scholar 

  • Woo HY, Park DI, Park H, Kim MK, Kim DH, Kim IS, Kim YJ (2009) Dual-priming oligonucleotide-based multiplex PCR for the detection of Helicobacter pylori and determination of clarithromycin resistance with gastric biopsy specimens. Helicobacter 14:22–28

    PubMed  Article  CAS  Google Scholar 

  • Yilmaz Ö, Demiray E (2007) Clinical role and importance of fluorescence in situ hybridization method in diagnosis of H. pylori infection and determination of clarithromycin resistance in H pylori eradication therapy. World J Gastroenterol 13:671–675

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors offer their thanks and appreciation to the Islamic Azad University, Jahrom and Shahrekord branches for their executive protection of this project.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Mohammad Kargar.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kargar, M., Doosti, A. & Ghorbani-Dalini, S. Detection of four clarithromycin resistance point mutations in Helicobacter pylori: comparison of real-time PCR and PCR-RFLP methods. Comp Clin Pathol 22, 1007–1013 (2013). https://doi.org/10.1007/s00580-012-1519-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00580-012-1519-1

Keywords

  • Clarithromycin resistant
  • Helicobacter pylori
  • PCR-RFLP
  • Real-time PCR