Lab Med Online.  2011 Jan;1(1):19-25.

Evaluation of the Performance of GenoType(R) MTBDRplus Assay for Rapid Detection of Multi-drug Resistant Mycobacterium tuberculosis in Sputum Specimens

• Jeong TD
• An D
• Sung H
• Chi HS
• Kim MN
• Shim TS

Abstract

BACKGROUND
GenoType(R) MTBDRplus assay (Hain Lifescience, Germany) enables detection of the mutations prevalent in rpoB, katG, and inhA genes and identification of Mycobacterium tuberculosis complex (MTB). We evaluated the performance of the MTBDRplus assay in detecting multidrug resistant M. tuberculosis in sputum specimens by directly comparing it to the performance of conventional drug susceptibility testing (DST) with M. tuberculosis culture isolates.
METHODS
From December 2007 to July 2008, 40 patients with acid-fast bacilli (AFB) smear-positive and AFB culture-positive sputa, including 19 patients with rifampin (RIF)- or isoniazid (INH)-resistant MTB isolates, were enrolled. The MTBDRplus assay was performed using DNA extracted from respiratory specimens. DST of the culture isolates was performed using an absolute concentration method.
RESULTS
The result of the AFB smear test was +/-1 for 7 specimens, +1 for 8 specimens, +2 for 9 specimens, +3 for 9 specimens, and +4 for 7 specimens. The MTBDRplus assay revealed that 37 of the 40 specimens were positive for an MTB-specific band, 12 specimens were RIF-resistant, and 16 specimens were INH-resistant. The rpoB S531L mutation was detected in 58.3% of the RIF-resistant specimens, and the katG S315T1 and inhA C15T mutations were detected in 56.3% and 31.3% of the INH-resistant specimens, respectively. Compared to the sensitivity and specificity of DST, both sensitivity and specificity of MTBDRplus assay for RIF resistance were 100%, and the corresponding values for INH resistance were 82.4% and 90.0%. Discrepant MTBDRplus assay and DST results were obtained in 3 INH-resistant isolates without mutation and 2 INH-susceptible isolates with katG S315T1 and inhA C15T mutations.
CONCLUSIONS
The MTBDRplus assay can be applied for AFB smear-positive specimens with positivity +/- to 4+. The assay was reliable for predicting the RIF resistance of culture isolates, but DST was required for confirming INH resistance.

Keyword

GenoType(R) MTBDRplus assay; Rifampin; Isoniazid; Multi-drug resistant Mycobacterium tuberculosis

MeSH Terms

DNA
Humans
Isoniazid
Mycobacterium
Mycobacterium tuberculosis
Rifampin
Sensitivity and Specificity
Sputum
Tuberculosis
DNA
Isoniazid
Rifampin

Reference

1. Korea Centers for Disease Control and Prevention. Annual report on the notified tuberculosis patients in Korea 2009. 2010. Seoul: Korea Centers for Disease Control and Prevention.

2. Pablos-Mendez A, Raviglione MC, Laszlo A, Binkin N, Rieder HL, Bustreo F, et al. Global surveillance for antituberculosis-drug resistance, 1994-1997. World Health Organization-International Union against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance. N Engl J Med. 1998. 338:1641–1649.

3. Centers for Disease Control and Prevention (CDC). Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs--worldwide, 2000-2004. MMWR Morb Mortal Wkly Rep. 2006. 55:301–305.

4. Velayati AA, Masjedi MR, Farnia P, Tabarsi P, Ghanavi J, Ziazarifi AH, et al. Emergence of new forms of totally drug-resistant tuberculosis bacilli: super extensively drug-resistant tuberculosis or totally drug-resistant strains in iran. Chest. 2009. 136:420–425.

5. Wright A, Zignol M, Van Deun A, Falzon D, Gerdes SR, Feldman K, et al. Epidemiology of antituberculosis drug resistance 2002-07: an updated analysis of the Global Project on Anti-Tuberculosis Drug Resistance Surveillance. Lancet. 2009. 373:1861–1873.

6. Diagnostic Standards and Classification of Tuberculosis in Adults and Children. This official statement of the American Thoracic Society and the Centers for Disease Control and Prevention was adopted by the ATS Board of Directors, July 1999. This statement was endorsed by the Council of the Infectious Disease Society of America, September 1999. Am J Respir Crit Care Med. 2000. 161:1376–1395.

8. Pfyffer GE. Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, editors. Mycobacterium: general characteristics, laboratory detection, and staining procedures. Manual of clinical microbiology. 2007. 9th ed. Washington DC: ASM press;1223–1236.

9. Chang CL, Park TS, Kim MN, Lee NY, Lee HJ, Suh JT, et al. Survey on changes in mycobacterial testing practices in Korean Laboratories. Korean J Clin Microbiol. 2001. 4:108–114.

10. Pai M, Kalantri S, Dheda K. New tools and emerging technologies for the diagnosis of tuberculosis: part II. Active tuberculosis and drug resistance. Expert Rev Mol Diagn. 2006. 6:423–432.

11. Hillemann D, Rusch-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. 45:2635–2640.

12. 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. 43:3699–3703.

13. Chang C, Jeong J. Korean Society for Laboratory Medicine. Mycobacteria, Nocardia and other aerobic Actinomycetes. Laboratory Medicine 4th ed. 2009. Seoul: E*PUBLIC;504–505.

14. Akpaka PE, Baboolal S, Clarke D, Francis L, Rastogi N. Evaluation of methods for rapid detection of resistance to isoniazid and rifampin in Mycobacterium tuberculosis isolates collected in the Caribbean. J Clin Microbiol. 2008. 46:3426–3428.

15. Brossier F, Veziris N, Jarlier V, Sougakoff W. Performance of MTBDR plus for detecting high/low levels of Mycobacterium tuberculosis resistance to isoniazid. Int J Tuberc Lung Dis. 2009. 13:260–265.

16. Causse M, Ruiz P, Gutierrez JB, Zerolo J, Casal M. Evaluation of new GenoType MTBDRplus for detection of resistance in cultures and direct specimens of Mycobacterium tuberculosis. Int J Tuberc Lung Dis. 2008. 12:1456–1460.

17. Huang WL, Chen HY, Kuo YM, Jou R. Performance assessment of the GenoType MTBDRplus test and DNA sequencing in detection of multidrug-resistant Mycobacterium tuberculosis. J Clin Microbiol. 2009. 47:2520–2524.

18. Huyen MN, Tiemersma EW, Lan NT, Cobelens FG, Dung NH, Sy DN, et al. Validation of the GenoType MTBDRplus assay for diagnosis of multidrug resistant tuberculosis in South Vietnam. BMC Infect Dis. 2010. 10:149.

19. Lacoma A, Garcia-Sierra N, Prat C, Ruiz-Manzano J, Haba L, Roses 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. 46:3660–3667.

20. Lin HH, Kim HY, Yun YJ, Park CG, Kim BJ, Park YK, et al. Mutations of katG and inhA in MDR M. tuberculosis. Tuberc Respir Dis. 2007. 63:128–138.

21. Barnard M, Albert H, Coetzee G, O'Brien R, Bosman ME. Rapid molecular screening for multidrug-resistant tuberculosis in a high-volume public health laboratory in South Africa. Am J Respir Crit Care Med. 2008. 177:787–792.

22. Nikolayevskyy V, Balabanova Y, Simak T, Malomanova N, Fedorin I, Drobniewski F. Performance of the Genotype MTBDRPlus assay in the diagnosis of tuberculosis and drug resistance in Samara, Russian Federation. BMC Clin Pathol. 2009. 9:2.

23. Anek-Vorapong R, Sinthuwattanawibool C, Podewils LJ, McCarthy K, Ngamlert K, Promsarin B, et al. Validation of the GenoType MTBDRplus assay for detection of MDR-TB in a public health laboratory in Thailand. BMC Infect Dis. 2010. 10:123.

24. Telenti A, Imboden P, Marchesi F, Lowrie D, Cole S, Colston MJ, et al. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet. 1993. 341:647–650.

25. Miotto P, Piana F, Cirillo DM, Migliori GB. Genotype MTBDRplus: a further step toward rapid identification of drug-resistant Mycobacterium tuberculosis. J Clin Microbiol. 2008. 46:393–394.

26. Ahmad S, Mokaddas E. Recent advances in the diagnosis and treatment of multidrug-resistant tuberculosis. Respir Med. 2009. 103:1777–1790.

27. Hazbon MH, Brimacombe M, Bobadilla del Valle M, Cavatore M, Guerrero MI, Varma-Basil M, et al. Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2006. 50:2640–2649.

28. Abe C, Kobayashi I, Mitarai S, Wada M, Kawabe Y, Takashima T, et al. Biological and molecular characteristics of Mycobacterium tuberculosis clinical isolates with low-level resistance to isoniazid in Japan. J Clin Microbiol. 2008. 46:2263–2268.

29. Hofmann-Thiel S, van Ingen J, Feldmann K, Turaev L, Uzakova GT, Murmusaeva G, et al. Mechanisms of heteroresistance to isoniazid and rifampin of Mycobacterium tuberculosis in Tashkent, Uzbekistan. Eur Respir J. 2009. 33:368–374.

30. Rinder H, Mieskes KT, Loscher T. Heteroresistance in Mycobacterium tuberculosis. Int J Tuberc Lung Dis. 2001. 5:339–345.