Meryem ÇETİN, Murat GÜNAYDIN, Ahmet SANIÇ

Mycobacterium tuberculosis kompleksinde izoniazid ve rifampisin direncinin real-time PCR ile hızlı tanısı

Otuz izoniazide dirençli, 30 rifampisine dirençli, 21 hem izoniazid hem de rifampisine dirençli ve kontrol grubu olarak her iki ilaca duyarlı 30 Mycobacterium tuberculosis suşu radyometrik BACTEC metodu kullanılarak belirlenmiştir. KatG and rpoB sensor probları kullanılarak, M.tuberculosis mutasyonlarının varlığı real-time PCR metodu ile incelenmiştir. İzoniazid için 17 izolatta prob Tm aralığı 66.98-67.70°C ve 6 izolatta prob Tm aralığı 68.19-68.90°C olarak belirlenmiştir. Toplam 23 izolatta dirence sebep olan mutasyonlar saptanmıştır. Rifampisin için, 9 izolatta prob Tm aralığı 65.3-66.70°C, 22 izolatta prob Tm aralığı 57.70-58.40°C ve 4 suşta bu Tm aralıklarının her ikisi gösterilmiştir. Toplam 27 suşta dirence sebep olan mutasyonlar belirlenmiştir. Real-time PCR metodu izoniazid direncini % 76.7 olarak belirlemiştir. BACTEC ve real-time PCR metodları arasında istatistiksel olarak anlamlı bir fark saptanmıştır (p=0.005). Real-time PCR ile rifampisin direnci % 90.0 olarak belirlenmiş, BACTEC ve real-time PCR arasında istatistiksel olarak anlamlı bir fark saptanmamıştır (p=0.076). Bu sonuçlar real-time PCR metodunun rutin laboratuvarda M.tuberculosis komplekste ilaç direncini belirlemede hızlı, basit ve güvenilir bir metod olduğunu göstermiştir.

Anahtar Kelimeler:

İzoniazid, Rifampin, Mycobacterium tuberculosis, Mutasyon, Polimeraz zincir reaksiyonu

Rapid detection of isoniazid and rifampicin resistance associated mutations in Mycobacterium tuberculosis complex by real-time PCR

Susceptibilities of 30 isoniazid-resistant, 30 rifampicin-resistant and 21 isoniazid and rifampisin-resistant Mycobacterium tuberculosis isolates as well as 30 isolates susceptible to the both drugs used as control group were determined by radyometric BACTEC method. Using katG and rpoB sensor probes, presence of mutations was investigated in M.tuberculosis by real-time PCR method. For isoniazid, prob Tm range of 66.98-67.70°C in 17 isolates and prob Tm range of 68.19-68.90°C in 6 isolates were found. Resistance-causing mutations were detected in a total of 23 isolates. For rifampicin, prob Tm range of 65.3-66.70°C in 9 isolates, prob Tm range of 57.70-58.40°C in 22 isolates and both of these Tm ranges in 4 isolates were determined. Mutations causing resistance were detected in a total of 27 isolates. By real-time PCR method isoniazid mutations were detected as 76.7 %. There was a statistically significant difference between BACTEC and real-time PCR methods (p=0.005). On the other hand, 90.0 % of rifampicin resistance were detected by real-time PCR, which indicated a statistically insignificant difference between BACTEC and real-time PCR (p=0.076). All these results showed that real-time PCR method is a fast, simple and accurate method for determining the drug resistance in M.tuberculosis complex in routine laboratories.

Keywords:

Isoniazid, Rifampin, Mycobacterium tuberculosis, Mutation, Polymerase Chain Reaction,

PDF

___

1. Bernard PS, Reiser A, Pritham GH: Mutation detection fluorescent hybridization probe melting curves, “Maver S, Wittwer C, Nekagawara K (eds): Rapid Cylce Real-Time PCR. Methods and Applications” kitabında s.11-9, Springer Verlag, Berlin-Heidelberg (2001).
2. Cole ST: Mycobacterium tuberculosis: drug resistance mechanisms, Trends Microbiol 1994;2(10):411-5.
3. Çalışır HC, Şipit T, Öğretensoy M: Tüberküloz: Tanı ve tedavisi, Tüberküloz ve Toraks 1998;146:81-9.
4. Daniel TM: The history of tuberculosis, Respir Med 2006;100(11):1862-70.
5. Drobniewski FA, Wilson SM: The rapid diagnosis of isoniazid and rifampicin resistance in Mycobacterium tuberculosis-a molecular story, J Med Microbiol 1998;47(3):189-96.
6. Gonzales N, Torres MJ, Anzar J, Palomares JC: Molecular analysis of rifampin and isoniazid resistance of Mycobacterium tuberculosis clinical isolates in Seville, Spain, Tuber Lung Dis 1999;79(3):187-90.
7. Kiepiela P, Bishop KS, Smith AN, Roux L, York DF: Genomic mutations in the katG, inhA and and aphC genes are useful for the prediction of isoniazid resistance in Mycobacterium tuberculosis isolates from Kwazulu Natal, South Africa, Tuber Lung Dis 2000;80(1):47-56.
8. Kocabaş A: Akciğer tüberkülozu, “Topçu AW, Söyletir G, Do¤anay M (eds): infeksiyon Hastalıkları” kitabında s.396-443, Nobel Tıp Kitabevi, İstanbul (1996).
9. Kocagoz T, Saribas Z, Alp A: Rapid determination of rifampin resistance in clinical isolates of Mycobacterium tuberculosis by Real-Time PCR, J Clin Microbiol 2005;43(12):6015-9.
10. Musser JM: Antimicrobial agent resistance in mycobacteria molecular genetic insights, Clin Microbiol Rev 1995;8(4):496-514.
11. Musser JM, Kapur V, Williams DL, Kreiswirth BN, van Soolingen D, van Embden JD: Characterization of the catalase-peroxidase gene (katG) and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance, J Infect Dis 1996;173(1):196-202.
12. Ramaswamy S, Musser JM: Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update, Tuber Lung Dis 1998;79(1):3-29.
13. Sacchettini JC, Blanchard JS: The structure and function of the isoniazid target in M.tuberculosis, Res Microbiol 1996;147(1-2):36-43.
14. Somoskovi A, Parsons L, Salfinger M: The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis, Respir Res 2001;2(3):164-8.
15. Telenti A, Honore N, Bernasconi C et al: Genotipic assesment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level, J Clin Microbiol 1997;35(3):719-23.
16. Torres M, Criado A, Palomares JC, Aznar J: Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance-assotiated mutations in Mycobacterium tuberculosis, J Clin Microbiol 2000;38(9):3194-9.
17. Wadsword Center: Clinical Mycobacteriology Lab. Procedures, New York State Dept. of Health, New York (1996).
18. Walsh PS, Metzger DA, Higuchi R: Chelex 100 as a medium for simple extraction of DNA for PCRbased typing from forensic material, Biotechniques 1991;10(4):506-13.
19. Zhang Y, Heym B, Allen B, Young D, Cole S: The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis, Nature 1992;358(6387):591-3.