Hülya ŞİMŞEK, Güven URAZ

Comparison of rifampicin drug susceptibility determined by conventional culture method and Pcr-heteroduplex analysis in Mycobacterium tuberculosis strains

Mycobacterium tuberculosis'in (MTB) antitüberküloz ilaçlara karşı direnç geliştirmesi büyük ölçüde halk sağlığını tehdit etmektedir. Son zamanlarda, MTB'deki rifampisin direncinin yüksek seviyede olması nedeniyle, tedavisi zorlaşmaktadır. Bu çalışmada, yaklaşık 2 yıl boyunca (1997-1998) Ankara, Atatürk Göğüs Hastalıkları ve Göğüs Cerrahisi Merkezi laboratuvarından izole edilen, kültürü pozitif izolatlardan 127 rifampisin dirençli ve 33 rifampisin duyarlı suşlar rasgele seçilmiştir. Tüberkülozdan şüphelenilen hastalardan toplanan klinik örneklerden asido resistan basil (ARB)çalışılmıştır ve MTB saptamak için kültüre alınmıştır. Rifampisin direnciyle ilgili gen bölgesini çoğaltmak için PCR kullanılmıştır. rpoB geninin çoğaltılan 305 baz çiftlik parçası için MTB'deki rifampisin direnç fenotipini hızlı saptamak amacıyla Heterodupleks analizi uygulanmıştır. Klasik kültür yöntemine göre seçilmiş 127 rifampisin dirençli ve 33 rifampisin duyarlı izolatlar tekrar PCR-Heterodupleks analizi ile test edilmişlerdir. PCR-Heterodupleks analizine göre 160 M. tuberculosis suşlarından 105'i rifampisin dirençli ve 55 suş ise rifampisin duyarlı bulunmuştur. Klasik kültür metoduyla saptanan 127 rifampisin dirençli izolattan 101'i Heterodupleks analizi ile uyumluydu, fakat 26 izolat uyum göstermemiştir. Dört suş klasik kültür yöntemiyle rifampisine duyarlı olarak saptandığı halde, heterodupleks analizi ile rifampisine dirençli bulunmuşlardır. Heterodupleks analizi ile rifampisin dirençli olarak gözlenmeyen 26 suşta çoğaltılan parçanın dışında bir mutasyon, yada klasik kültür metodu ile saptanan direnç sonuçlarında bir hata olabileceği düşünülmüştür. Sonuç olarak, yine de PCR-Heterodupleks analizi nispeten pahalı bir teknik olmasına rağmen, rifampisin direncini saptamak için hızlı ve güvenli bir metottur.

Mycobacterium tuberculosis suşlarında klasik kültür metodu ve Pcr-heteroduplex analizi ile saptanan rifampisin ilaç duyarlılığının karşılaştırılması

Resistance of Mycobacterium tuberculosis (MTB) to antituberculous drugs has been threatening the public-health grossly. Recently, because of high-level of resistance to rifampicin in MTB, treatment is becoming impossible. In this study, 127 rifampicin resistant and 33 rifampicin susceptible strains were randomly chosen from tuberculosis culture-positive isolates, isolated in the laboratory of the Ataturk Chest Diseases and Chest Surgery Center in Ankara, Turkey, over a 2-year period (1997 to 1998). Clinical samples collected from patients with suspected tuberculosis were studied for acid-fast bacilli stain (AFBS) and cultured for detecting MTB. PCR was used to amplify genetic loci associated with rifampicin resistance. The amplified 305-bp fragment of the rpoB gene was applied to Heteroduplex assay for rapid detection of rifampicin resistance phenotype in MTB. The 127 rifampicinresistant and 33 rifampicin-susceptible isolates, selected according to conventional culture method, were tested again by PCR-Heteroduplex analysis. According to PCR-Heteroduplex analysis, 105 isolates of 160 M. tuberculosis were rifampicin resistant, 55 strains were rifampicin susceptible. Of 127 rifampicin-resistant isolates detected by conventional culture method, 101 had concordance with Heteroduplex analysis, whereas 26 isolates did not. While 4 strains were detected as rifampicin susceptible by conventional culture technique, they were rifampicin resistant by Heteroduplex analysis. It is thought that there might be a mutation out of fragment amplified, in 26 strains which were not observed as rifampicin resistant by Heteroduplex analysis; or a mistake in the result of resistance defined by classic culture method. In conclusion, however, PCR-Heteroduplex analysis is a rapid and reliable method to detect rifampicin resistance, although it is a relatively expensive technique.

PDF

___

1. Snider DF, Raviglione M, Kochi A. Global burden of tuberculosis: In Tuberculosis, Pathogeneses, Protection and Control. Bloom BR (Ed.), ASM Press, Washington D.C., 1994; 3-11.
2. Heym B, Honore N, Truffot-Pernot C, et. al. Implications of multidrug resistance for the future of short-course chemotherapy of tuberculosis: a molecular study. Lancet 1995;344: 293-298.
3. Mitchison DA. The action of antituberculosis drugs in short-course chemotherapy. Tubercle 1985; 66: 219-226.
4. Jindani A, Aber VR, Edward EA, et. al. The early bactericidal activity of drugs in patients with pulmonary tuberculosis. Am. Rev. Respir. Dis 1980; 121: 239-249
5. Calisir HC, Yilmaz H, Sarioglu N, et. al. The effects of initial isoniasid and rifampicin resistance to the prognosis of tuberculosis. Solunum Hastaliklari 1997; 8: 217-224
6. Harrow EM, Alcahe IC, Rruiz ILR, et. al. Treatment of drug sensitive and drug resistant tuberculosis in Guatamala. Chest 1994;106, suppl. 113
7. Mitchison DA, Nunn AJ. Influence of initial drug resistance on the response to short course chemotherapy of pulmonary tuberculosis. Am. Rev. Respir. Dis 1986;133: 423-430
8. Balci I, Dikensoy BA, Filiz A. Drug-resistant tuberculosis of the University Hospital in Gaziantep, Southern Turkey. J. Int. Med. Res 2000;28: 300-306 Bengisun JS, Karnak D, Palabiyikoglu I, Saygun N. Mycobacterium tuberculosis drug resistance in Turkey, 1976-97. Scand. J. Infect. Dis 2000;32: 507-510
10. Hart CA, Beeching NJ, Duerden BI. Tuberculosis into the next centurry. J. Med. Microbiol 1996;44: 1-34.
11. Heifets LB. Expedited detection of drug resistance in tuberculosis patients, Ann. Emerg. Med 1994;24: 457-461
12. Heifets LB. Mycobacteriology laboratory, Clin. Chest Med 1997;18: 35-53
13. Pfaller MA. Application of new technology to the detection, identification and antimicrobial susceptibility testing of mycobacteria. Am. J. Clin. Pathol 1994; 101: 329-337
14. Wellstood S. Diagnostic mycobacteriology: current challenges and technologies. Lab. Med 1993; 24: 357-361
15. Ellner JJ, Hinman S, Dooley SW, et. al. Tuberculosis symposium: emerging problems and promise. Journal of Infection Disease 1993;168: 537-551
16. Musser JM. Antimicrobial agent resistance in mycobacteria: molecular genetic insights. Clin. Microbiol. Rev . 1995; 8: 496-514
17. Isenberg HD. Digestion-decontamination procedures, In: Isenberg H.D (Ed), Clinical Microbiology Procedures Handbook Volume 1. Washington DC.1992; 3.4.2.
18. Wilson SM, McNerney R, Nye PM, et. al. Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis. Journal of Clinical Microbiology .1993; 31: 776-782
19. Nolte FS, Metchock B. Mycobacterium, Manual of Clinical Microbiology. Murrey PR, Baron EJO, Pfaller MA, Tenover FC, Yolken RH, Sixth Edition, ASM Press, Washington, D. C. 1995; 400-437
20. Kent PT, Kubica GP. Public Health Mycobacteriology: a Guide for the Level III Laboratory, Centers for Disease Control, Atlanta 1985
21. Koneman EW, Allen SD, Janda WM, et. al. Mycobacteria, In Diagnostic Microbiology, 5th Ed. Philadelphia, New York. 1997;703-756
22. Warren NG, Boby BA, Dalton HP. An improved reagent for mycobacterial nitrate reductase tests. J. Clin. Microbiol 1983;18:546-549
23. Canetti G, Froman S, Grosset J. Mycobacteria: Laboratory methods for testing drug sensitivity and resistance. Bulletin of World Health Organization. 1997
24. Gumuslu F, Ceyhan I, Kocagoz T, et. al. The guidebook for mycobacteria laboratory, Republic of Turkey, Ministry of Health, Refik Saydam Hygiene Center, 1st Ed. Ankara, Turkey 1998
25. Miller LP, Crawford JT, Shinnick FM. The rpoB gene of Mycobacterium tuberculosis. Antimicrobial Agents of Chemotherapy 1994;38: 805-811.
26. Drobniewski FA, Wilson SM. The rapid diagnosis of isoniazid and rifampicin resistance in Mycobacterium tuberculosis-a molecular story. J. Medical Microbiol 1998; 47: 189-196
27. Williams DL, Waguespack C, Eisenach K, et. al. Characterization of rifampicin resistance in pathogenic mycobacteria. Antimicrobial Agents of Chemotherapy 1994;38: 2380-2386
28. Williams DL, Limbers C, Spring L, et. al. PCR-Heteroduplex detection of rifampin-resistant Mycobacterium tuberculosis. PCR protocols for emerging infectious diseases. In D.H. Persing (ed.). ASM Press, Washington, DC. 1997; 122-129
29. Williams DL, Spring L, Gillis T P, et. al. Evaluation of a polymerase chain reaction-based universal heteroduplex generator assay for direct detection of rifampin susceptibility of Mycobacterium tuberculosis from sputum specimens. Clinical Infection Diseases 1998;26: 446-450
30. Williams DL. Molecular methods to determine drug resistance in pathogenic Mycobacteria, Bulletin of Microbiology, 1st National Molecular and Diagnostic Microbiology Congress Special Issue 2000; 34: 23-39
31. Kim BJ, Kim SY, Park BH, et. al. Mutations in the rpoB gene of Mycobacterium tuberculosis that interfere with PCR-single-strand conformation polymorphism analysis for rifampin susceptibility testing. J. Clin. Microbiol 1997;35: 492-494