Real-Time PCR Assay Using Allele-Specific TaqMan probe for Detection of Clarithromycin Resistance and Its Point Mutations in Helicobacter Pylori

Document Type : Original Article (s)

Authors

1 Associate Professor, Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran.

2 Department of Microbiology, Jahrom Branch, Young Researcher's Clube, Islamic Azad University, Jahrom, Iran.

3 Assistant Professor, Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.

4 Assistant Professor, Biotechnology Research Center, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran

Abstract

Background: Helicobacter pylori is a fastidious microorganism and therefore standard phenotypic susceptibility tests can take at least 10-14 days. Molecular based diagnostic assays offer an alternative approach to obtain susceptibilities to antibiotics and detection of point mutations with greater accuracy. The aim of this study was the assessment of Clarithromycin resistance and its point mutations by using real-time PCR assay.Methods: This cross-sectional descriptive study was performed on 200 gastric biopsy specimens obtained from patients undergoing upper gastrointestinal endoscopy at Hajar hospital in Shahrekord. Initially Helicobacter pylori were identified by rapid urease test (RUT) and polymerase chain reaction (PCR). Then clarithromycin resistance and its point mutations were evaluated by using specific probes and real-time PCR technique.Finding: Of total samples, 164 (82%) were Helicobacter pylori positive. Overall, a clarithromycin susceptible strains were detected in 105 (64.02%) patients and resistance strains were detected in 59 (35.98%) which were identified as 4 (2.44%) A 2144G, 26 (15.85%) A2143G, 15 (9.15%) A2143C, and 20 (12.19%) A2142G point mutations. Genotype of 5 (8.47%) strains was not detected. Purely resistant strains were detected in 38 (23.17%), while heteroresistant were found in the remaining 16 cases (9.76%).Conclusion: Results showed that real-time PCR assay has high accuracy to simultaneously identify Helicobacter pylori and clarithromycin resistance types directly in gastric biopsy specimens in short time.      

Keywords


  1. Goldman RC, Zakula D, Flamm R, Beyer J, Capobianco J. Tight binding of clarithromycin, its 14-(R)-hydroxy metabolite, and erythromycin to Helicobacter pylori ribosomes. Antimicrob Agents Chemother 1994; 38(7): 1496-500.
  2. Lottspeich C, Schwarzer A, Panthel K, Koletzko S, Russmann H. Evaluation of the novel Helico-bacter pylori ClariRes real-time PCR assay for detection and clarithromycin susceptibility test-ing of H. pylori in stool specimens from symp-tomatic children. J Clin Microbiol 2007; 45(6): 1718-22.
  3. Chisholm SA, Owen RJ, Teare EL, Saverymuttu S. PCR-based diagnosis of Helicobacter pylori infection and real-time determination of clar-ithromycin resistance directly from human gas-tric biopsy samples. J Clin Microbiol 2001; 39(4): 1217-20.
  4. Taneike I, Suzuki K, Nakagawa S, Yamamoto T. Intrafamilial spread of the same clarithromy-cin-resistant Helicobacter pylori infection con-firmed by molecular analysis. J Clin Microbiol 2004; 42(8): 3901-3.
  5. Lee JH, Shin JH, Roe IH, Sohn SG, Lee JH, Kang GH, et al. Impact of clarithromycin resistance on eradication of Helicobacter pylori in infected adults. Antimicrob Agents Chemother 2005; 49(4): 1600-3.
  6. Liu Z, Shen J, Zhang L, Shen L, Li Q, Zhang B, et al. Prevalence of A2143G mutation of H. pylori-23S rRNA in Chinese subjects with and without clarithromycin use history. BMC Microbiol 2008; 8: 81.
  7. Lascols C, Lamarque D, Costa JM, Copie-Bergman C, Le Glaunec JM, Deforges L, et al. Fast and accurate quantitative detection of Hel-icobacter pylori and identification of clarithro-mycin resistance mutations in H. pylori isolates from gastric biopsy specimens by real-time PCR. J Clin Microbiol 2003; 41(10): 4573-7.
  8. van Doorn LJ, Glupczynski Y, Kusters JG, Megraud F, Midolo P, Maggi-Solca N, et al. Ac-curate prediction of macrolide resistance in Heli-cobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: multicenter validation study. Antimicrob Agents Chemother 2001; 45(5): 1500-4.
  9. Pina M, Occhialini A, Monteiro L, Doermann HP, Megraud F. Detection of point mutations as-sociated with resistance of Helicobacter pylori to clarithromycin by hybridization in liquid phase. J Clin Microbiol 1998; 36(11): 3285-90.
  10. Ribeiro ML, attos RG, endonca S, edrazzoli Jr J. Quantitative real-time PCR for the clinical detec-tion of Helicobacter pylori. Genetics and Molec-ular Biology 2007; 30(2): 431-4.
  11. Schabereiter-Gurtner C, Hirschl AM, Dragosics B, Hufnagl P, Puz S, Kovach Z, et al. Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy speci-mens. J Clin Microbiol 2004; 42(10): 4512-8.
  12. Kalach N, Benhamou PH, Campeotto F, Ber-geret M, Dupont C, Raymond J. Clarithromycin resistance and eradication of Helicobacter pylori in children. Antimicrob Agents Chemother 2001; 45(7): 2134-5.
  13. Oleastro M, Menard A, Santos A, Lamouliatte H, Monteiro L, Barthelemy P, et al. Real-time PCR assay for rapid and accurate detection of point mutations conferring resistance to clar-ithromycin in Helicobacter pylori. J Clin Micro-biol 2003; 41(1): 397-402.
  14. Russmann H, Adler K, Haas R, Gebert B, Koletzko S, Heesemann J. Rapid and accurate determination of genotypic clarithromycin re-sistance in cultured Helicobacter pylori by fluo-rescent in situ hybridization. J Clin Microbiol 2001; 39(11): 4142-4.
  15. van der Ende A, van Doorn LJ, Rooijakkers S, Feller M, Tytgat GN, Dankert J. Clarithromycin-susceptible and -resistant Helicobacter pylori iso-lates with identical randomly amplified poly-morphic DNA-PCR genotypes cultured from sin-gle gastric biopsy specimens prior to antibiotic therapy. J Clin Microbiol 2001; 39(7): 2648-51.
  16. Fontana C, Favaro M, Pietroiusti A, Pistoia ES, Galante A, Favalli C. Detection of clarithromy-cin-resistant Helicobacter pylori in stool samples. J Clin Microbiol 2003; 41(8): 3636-40.
  17. Prazeres MP, De Magalhaes Queiroz DM, Cam-pos Barbosa DV, Aguiar RG, Nogueira ME, San-tos A, et al. Helicobacter pylori primary re-sistance to metronidazole and clarithromycin in Brazil. Antimicrob Agents Chemother 2002; 46(6): 2021-3.
  18. Godoy AP, Ribeiro ML, Benvengo YH, Vitiello L, Miranda MC, Mendonca S, et al. Analysis of antimicrobial susceptibility and virulence factors in Helicobacter pylori clinical isolates. BMC Gas-troenterol 2003; 3: 20.
  19. Torres J, Camorlinga-Ponce M, Perez-Perez G, Madrazo-De la Garza A, Dehesa M, Gonzalez-Valencia G, et al. Increasing multidrug resistance in Helicobacter pylori strains isolated from chil-dren and adults in Mexico. J Clin Microbiol 2001; 39(7): 2677-80.
  20. Alarcon T, Vega AE, Domingo D, Martinez MJ, Lopez-Brea M. Clarithromycin resistance among Helicobacter pylori strains isolated from chil-dren: prevalence and study of mechanism of re-sistance by PCR-restriction fragment length pol-ymorphism analysis. J Clin Microbiol 2003; 41(1): 486-99.
  21. Stone GG, Shortridge D, Versalovic J, Beyer J, Flamm RK, Graham DY, et al. A PCR-oligonucleotide ligation assay to determine the prevalence of 23S rRNA gene mutations in clar-ithromycin-resistant Helicobacter pylori. Antimi-crob Agents Chemother 1997; 41(3): 712-4.
  22. 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(1): 259-61.
  23. Kato M, Yamaoka Y, Kim JJ, Reddy R, Asaka M, Kashima K, et al. Regional differences in metronidazole resistance and increasing clar-ithromycin resistance among Helicobacter pylori isolates from Japan. Antimicrob Agents Chemother 2000; 44(8): 2214-6.
  24. Kumala W, Rani A. Patterns of Helicobacter pylori isolate resistance to fluoroquinolones, amoxicillin, clarithromycin and metronidazoles. Southeast Asian J Trop Med Public Health 2006; 37(5): 970-4.
  25. Kargar M, Baghernejad M, Doosti A. Evaluating the prevalence of A2142G, A2143G, and A2142C mutations in clarithromycin resistance Helicobacter pylori in Chaharmahal and Bakhtiari province, Iran. Journal of Kordestan University of Medical Sciences 2010; 14(4): 72-8.
  26. Marais A, Monteiro L, Occhialini A, Pina M, Lamouliatte H, Megraud F. Direct detection of Helicobacter pylori resistance to macrolides by a polymerase chain reaction/DNA enzyme immu-noassay in gastric biopsy specimens. Gut 1999; 44(4): 463-7.
  27. Maeda S, Yoshida H, Ogura K, Kanai F, Shirato-ri Y, Omata M. Helicobacter pylori specific nest-ed PCR assay for the detection of 23S rRNA mutation associated with clarithromycin re-sistance. Gut 1998; 43(3): 317-21.
  28. Matsuoka M, Yoshida Y, Hayakawa K, Fukuchi S, Sugano K. Simultaneous colonisation of Heli-cobacter pylori with and without mutations in the 23S rRNA gene in patients with no history of clarithromycin exposure. Gut 1999; 45(4): 503-7.
  29. Matsumura M, Hikiba Y, Ogura K, Togo G, Tsu-kuda I, Ushikawa K, et al. Rapid detection of mutations in the 23S rRNA gene of Helicobacter pylori that confers resistance to clarithromycin treatment to the bacterium. J Clin Microbiol 2001; 39(2): 691-5.