Tuba ÖZTÜRK, Buket CİCİOĞLU ARIDOĞAN, Emel SESLİ ÇETİN

MYCOBACTERİUM TUBERCULOSİS KOMPLEKSİ KLİNİK İZOLATLARINDA İZONİAZİD VE RİFAMPİSİN DİRENCİNİN HIZLI TANISI İÇİN ‘REVERSE BLOT HYBRIDIZATION ASSAY MYCOBACTERIUM TUBERCULOSIS DRUG RESISTANCE’ YÖNTEMİNİN ETKİNLİĞİNİN ARAŞTIRILMASI

Amaçİzoniazid (INH) ve rifampisin (RIF) dirençli Mycobacterium tuberculosis kompleksi klinik izolatlarının hızlı tespiti, erken ve uygun tedavinin planlanmasında enönemli basamaktır. ‘Reverse blot hybridization assay Mycobacterium tuberculosis drug resistance’ (REBA MTB-MDR), klinik izolatlarda rpoB, katG, inhA, ahpCgen mutasyonlarının hızlı tespiti için tasarlanan ve ticari olarak temin edilen reverse blot hibridizasyon esaslı bir DNA şerit testidir. Bu çalışmanın amacı M.tuberculosis kompleksi klinik izolatlarında INH ve RIF direnci ile ilişkili mutasyon tiplerinin REBA MTB-MDR testi ile belirlenmesi ve testin tanısal performansınınBACTEC MGIT 960 ile karşılaştırılmasıdır.Gereç ve YöntemÇalışma, 2008-2013 yılları arasında Süleyman Demirel Üniversitesi Tıp Fakültesi Mikrobiyoloji laboratuvarında çeşitli klinik örneklerden izole edilen 55 M. tuberculosis kompleks suşu ile yapıldı. Suşların primer anti tüberküloz ilaç duyarlılıkları BACTEC MGIT 960 sistemiyle belirlendikten sonra INH ve RIF direnci gen mutasyonları REBA MTB-MDR testi ile araştırıldı.BulgularÇalışılan 55 izolatın MGIT 960 sistemiyle 41’i (%74.5) primer anti tüberküloz ilaçlara duyarlı, 14’ü (%25.5) dirençli bulundu. REBA MTB-MDR, MGIT 960 ile karşılaştırılarak değerlendirildiğinde RIF direncini saptamada duyarlılık %25 (CI 4.55-69.94), özgüllük %100 (CI 92.13-100) olarak, INH direncini saptamada iseduyarlılık %22.2 (CI 6.32-54.74), özgüllük %97.5 (CI 87.12-99.56) olarak belirlendi.SonuçREBA MTB-MDR testinin, dirençli M. tuberculosis kompleksi klinik izolatlarında en yaygın görülen mutasyonların hızlı tespitinde ve uygun tedaviye başlamada hız kazandırması açısından faydalı olduğu görülmüştür. Ancak bu testin sık görülmeyen mutasyonları belirlemedeki kısıtlılığı ve ilaç direncine neden olabilecek başka mekanizmaların varlığı nedeni ile rutin klinik laboratuvar uygulamalarında tek başınadeğil, geleneksel duyarlılık testleri ile birlikte kullanılmasının daha doğru olacağı kanaatine varılmıştır.

INVESTIGATION OF THE EFFECTIVENESS OF ‘REVERSE BLOT HYBRIDIZATION ASSAY MYCOBACTERIUM TUBERCULOSIS DRUG RESISTANCE’ METHOD FOR THE RAPID DIAGNOSIS OF ISONIAZID AND RIFAMPICIN RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS COMPLEX CLINICAL ISOLATES

Objective Rapid diagnosis of isoniazid (INH) and rifampicin (RIF) resistant Mycobacterium tuberculosis complex isolates is the most important step in planning of early and appropriate treatment. ‘Reverse blot hybridization assay Mycobacterium tuberculosis drug resistance’ (REBA MTB-MDR) is a commercially available reverse blot hybridization-based DNA strip test designed for the rapid detection of rpoB, katG, inhA, ahpC gene mutations in clinical isolates. The aim of this study was to determine the mutation types associated with INH and RIF resistance in clinical isolates of M. tuberculosis complex by REBA MTB-MDR test and compare the diagnostic performance of the test with BACTEC MGIT 960. Materials and Methods The study was performed with 55 M. tuberculosis complex strains isolated from various clinical samples in the microbiology laboratory of Suleyman Demirel University Medical Faculty between 2008-2013. The primary anti-tuberculosis drug susceptibilities of the strains were determined by the BACTEC MGIT 960 system and then INH and RIF resistance gene mutations were investigated by REBA MTB-MDR test. Results Of the 55 isolates tested with MGIT 960 system, 41 74.5%) were determined as susceptible to primary anti-tuberculosis drugs and 14 (25.5%) were determined as resistant to primary anti-tuberculosis drugs. When the REBA MTB-MDR was compared with the MGIT 960, the sensitivity to detect RIF resistance was 25% (CI 4.55-69.94), specificity was 100% (CI 92.13-100), sensitivity to detect INH resistance was 22.2% (CI 6.32-54.74), while specificity was 97.5% (CI 87.12-99.56) for REBA MTB-MDR. Conclusions REBA MTB-MDR test was found to be useful in terms of rapid detection of the most common mutations in the resistant M. tuberculosis complex clinical isolates and accelerating the initiation of appropriate treatment. However, due to the limitations of this test in identifying uncommon mutations and the presence of other mechanisms that may cause drug resistance, it was concluded that it would be more appropriate to use this test with conventional susceptibility tests in routine clinical laboratory practice.

PDF

___

1. Türkiye’de Verem Savaşı 2017 Raporu [Internet]. Ankara; 2017. [cited 15 October 2018] Available from: https://www.saglik.gov.tr
2. CLSI. Laboratory Detection and Identification of Mycobacteria; Approved Guideline. Clinical and Laboratory Standards Institute; 2008.
3. Jain A, Dixit P. Multidrug-resistant to extensively drug resistant tuberculosis: what is next? J Biosci. 2008;33(4):605–16.
4. National Committee for Clinical Laboratory Standards: Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes; Approved Standard. Pennsylvania: NCCLS document M24-A; 2003.
5. Durmaz R. Tüberkülozda Hızlı Moleküler Tanı Testleri. ANKEM derg. 2012;26(2):72–81.
6. Cruciani M, Scarparo C, Malena M, Bosco O, Serpelloni G, Mengoli C. Meta-Analysis of BACTEC MGIT 960 and BACTEC 460 TB, with or without Solid Media, for Detection of Mycobacteria. J Clin Microbiol. 2004;42(5):2321–5.
7. Adjers-Koskela K, Katila M-L. Susceptibility testing with the manual mycobacteria growth indicator tube (MGIT) and the MGIT 960 system provides rapid and reliable verification of multidrug-resistant tuberculosis. J Clin Microbiol. 2003;41(3):1235–9.
8. Report of Expert Consultations on Rapid Molecular Testing to Detect Drug-. Page last reviewed Resistant Tuberculosis in the United.[Internet] Center for Diseases Control. 2011 [cited 15 October 2018]. Available from: http://www.cdc.gov/tb/topic/laboratory/ rapidmoleculartesting/default.htm.
9. Bang H, Park S, Hwang J, Jin H, Cho E, Kim DY, et al. Improved rapid molecular diagnosis of multidrug-resistant tuberculosis using a new reverse hybridization assay, REBA MTB-MDR. J Med Microbiol. 2011;60(Pt 10):1447–54.
10. Cho E, Shamputa IC, Kwak H-K, Lee J, Lee M, Hwang S, et al. Utility of the REBA MTB-Rifa® assay for rapid detection of rifampicin resistant Mycobacterium tuberculosis. BMC Infect Dis. 2013;13:478.
11. MolecuTech REBA MTB-MDR, User’s Manuel. Korea: YD Diagnostics; 2012.
12. Dean AG, Sullivan KM, Soe MM. OpenEpi: Open Source Epidemiologic Statistics for Public Health, Version.[Internet] [cited 2019 Apr 28 ]. Available from: http://www.OpenEpi.com, updated 2013/04/06
13. Miotto P, Piana F, Penati V, Canducci F, Migliori GB, Cirillo DM. Use of genotype MTBDR assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical strains isolated in Italy. J Clin Microbiol. 2006 ;44(7):2485–91.
14. Ramaswamy SV, Reich R, Dou S-J, Jasperse L, Pan X, Wanger A, et al. Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2003;47(4):1241–50.
15. Brossier F, Veziris N, Truffot-Pernot C, Jarlier V, Sougakoff W. Performance of the Genotype MTBDR Line Probe Assay for Detection of Resistance to Rifampin and Isoniazid in Strains of Mycobacterium tuberculosis with Low- and High-Level Resistance. J Clin Microbiol. 2006;44(10):3659–64.
16. Hillemann D, Weizenegger M, Kubica T, Richter E, Niemann S. Use of the genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis complex isolates. J Clin Microbiol. 2005;43(8):3699–703.
17. Cavusoglu C, Turhan A, Akinci P, Soyler I. Evaluation of the Genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates. J Clin Microbiol. 2006;44(7):2338–42.
18. Aslan G, Tezcan S, Emekdaş G. Mycobacterium tuberculosis kompleksi klinik izolatlarında rifampisin ve izoniazid direncinin hızlı tespitinde “Genotype MTBDR” testinin değerlendirilmesi. Mikrobiyol Bul. 2009;43: 217–26.
19. Hillemann D, Rüsch-Gerdes S, Richter E. Evaluation of the GenoType MTBDRplus assay for rifampin and isoniazid susceptibility testing of Mycobacterium tuberculosis strains and clinical specimens. J Clin Microbiol. 2007;45(8):2635–40.
20. Lacoma A, Garcia-Sierra N, Prat C, Ruiz-Manzano J, Haba L, Rosés S, et al. GenoType MTBDRplus assay for molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis strains and clinical samples. J Clin Microbiol. 2008;46(11):3660–7.
21. Keerataya Ngamlert, Efficiency of drug resistant Mycobacterium tuberculosis detection betweenXpert MTB/RIF and Lıne Probe Assay Using REBA MTB-MDR. J Med Tech Assoc Thailaland. 2016;44(1)
22. Havumaki J, Hillemann D, Ismail N, Omar SV, Georghiou SB, Schumacher SG, et al. Comparative accuracy of the REBA MTB MDR and Hain MTBDRplus line probe assays for the detection of multidrug-resistant tuberculosis: A multicenter, non-inferiority study. PLOS ONE. 2017;12(3):e0173804.