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Molecular diagnosis of drug resistant tuberculosis by a DNA array

Molecular diagnosis of drug resistant tuberculosis by a DNA array. Tsung Chain Chang ( 張長泉 ) College of Medicine, National Cheng Kung University, Tainan, Taiwan 7 th Asia-Pacific Biotech Congress July 13-15, 2015, Beijing, China. Tuberculosis (TB).

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Molecular diagnosis of drug resistant tuberculosis by a DNA array

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  1. Molecular diagnosis of drug resistant tuberculosisby a DNA array Tsung Chain Chang(張長泉) College of Medicine, National Cheng Kung University, Tainan, Taiwan 7th Asia-Pacific Biotech Congress July 13-15, 2015, Beijing, China

  2. Tuberculosis (TB) • TB is caused by Mycobacterium tuberculosis (MTB), a very slow growing Gram-positive bacillus. Mycobacterium tuberculosis complex (MTBC)

  3. Tuberculosis (TB) • A major global health problem • 1/3 of world’s population are carriers; most are latent • TB cases are decreasing , but drug-resistant TB is increasing • MDR-TB(multidrug-resistantTB) resistanttoat least rifampin andisoniazid. • XDR-TB(extensively drug-resistantTB) MDR-TBwith resistance to a fluoroquinolone and either capreomycin, amikacin, or kanamycin.

  4. In 2013, an estimated 9.0 million people developed TB and 1.5 million died from TB. Global tuberculosis report 2014, WHO. Taiwan Tuberculosis Control Report 2013, Taiwan CDC.

  5. Global (2013) : 3.5% Global tuberculosis report 2014, WHO. Taiwan Tuberculosis Control Report 2013, Taiwan CDC.

  6. Global (2013) : 20.5% Global tuberculosis report 2014, WHO. Taiwan Tuberculosis Control Report 2013, Taiwan CDC.

  7. Laboratory diagnosis Clinical specimens Timeline (weeks) 0 1~2 MGIT 7H11 LJ 5 Drug susceptibility test 8

  8. Mycobacterial Growth Indicator Tube system (MGIT, BD) Fluorescent on the bottom is sensitive to the presence of oxygen dissolved in the broth. As the oxygen is used by microorganisms, the fluorescence can be detected.

  9. GenoType MTBDRplus (Hain Lifescience GmbH, Nehren, Germany) • A line probe hybridization assay. • Detection of rifampin and isoniazid resistance • GenoType MTBDRsl assay

  10. Xpert MTB/RIF (Cepheid) In December 2010, WHO endorsed the Xpert MTB/RIF for use in TB endemic countries and declared it a major milestone for global TB diagnosis. • Results are obtained from unprocessed sputum samples in 90minutes http://www.finddiagnostics.org/media/press/ 090324.html

  11. Incubator 15min DNA extraction >> heminested PCR

  12. Xpert MTB/RIF (Cepheid) • Results are obtained from unprocessed sputum samples in 90minutes • Minimal biohazard and very little technical training required to operate. • This test was developed as an on-demand near patient technology which could be performed even in a doctor's office. • Co-developed by Professor David Alland at the University of Medicine and Dentistry of New Jersey, Cepheid Inc. and Foundation for Innovative New Diagnostics, with additional financial support from NIH.

  13. Xpert MTB/RIF (Cepheid) • Simultaneous detection of both MTBC and rifampicin resistance. • Detecting MTB — even in smear negative, culture positive specimens • The concessional price for a GeneXpert system is USD 17,000 for a 4 module instrument. The cost of a test cartridge in countries eligible for concessional pricing is USD 9.98. FIND Diagnostics. FIND. October 2013. Retrieved 6 April 2014.

  14. Performance of Xpert MTB/RIF • Pooled sensitivity of 88% and specificity of 98%. • The sensitivity of the MTB/RIF test on just 1 sputum sample was 92.2% for culture-positive TB; 98.2% for smear- and culture-positive cases; and 72.5% for smear-negative, culture-positive cases. • Sensitivity and higher specificity were slightly higher when 3 samples were tested. Steingart KR et al. 2013. Cochrane Database of Systematic Reviews 2013: DOI: 10.1002/14651858.CD009593.pub2. Boehme et al. N Engl J Med. 2010; 363:1005-1015.

  15. FIND Diagnostics. FIND. Retrieved 6 April 2014.

  16. Evaluation of the Cobas TaqMan MTB Test for Direct Detection of MTBCin Respiratory Specimens • The Cobas Amplicor MTB assay (Roche Diagnostics) is based on amplification of the 16S rRNA gene, followed by colorimetric detection of the amplicon by probe hybridization. • The Cobas TaqMan MTB Test is based on real-time PCR and is used for detection of MTBC in pulmonary specimens, including smear-(+) and -(-) specimens. Yang et al. 2011. J. Clin. Microbiol. 49:797-801.

  17. Evaluation of the Cobas TaqMan MTB Test for Direct Detection of MTBC • 1,093 samples (446 patients), including 118 AFB-(+) and 975 AFB-(-) specimens. • The sensitivity, specificity, PPV, and NPV were 91.5%, 98.7%, 91.5%, and 98.7%, respectively. • High sensitivity (79.5%) for detecting MTBC in AFB-(-) specimens, 35/44 AFB-(-) were positive. Yang et al. 2011. J. Clin. Microbiol. 49:797-801.

  18. Drug susceptibility testing (agar proportion method) No. of colonies on drug-containing medium % Resistant ×100 = No. of colonies on drug-free medium If 1%  resistance Results are reported at three weeks afterinoculation. INH 0.2 μg/ml control EMB 7.5 μg/ml RIF 1 μg/ml Medium : Middlebrook 7H10 agar medium. RIF: rifampin; INH: isoniazid; EMB: ethambutol CLSI M24-A, 2003.

  19. The limits of culture methods Drug susceptibility test (Second-line) Drug susceptibility test (First-line) Culture & identification • Many XDR-TB cases are not diagnosed since testing for resistance to 2nd-line drugs is notroutinely performed. Blaschitz et al., 2011 • Accurate diagnosis and early initiation of treatmentare important to reduce transmission and hinder the emergence of drug-resistant TB.Ajbani et al., 2011. Agar proportional method Molecular method

  20. Treatment of TB Susceptible case: 6-month treatment with 3-5 drugs of 1st-line MDR case: 18 to 24-month treatment with 2nd-line 陸等人, 2008.

  21. Development of an array To detect point mutations in 9 genes to predict drug resistance of M. tuberculosis

  22. 546 clinical isolates (204 patients) Discrepant analysis Agar proportional method Oligonucleotide array

  23. Conclusion • The array can effectively detect drug resistance to the 1st and 2nd-line anti-TB drugs, except CAP. • Similar results from positive liquid cultures (MGIT). • The array can detect exact mutations, thus has epidemiology value. • The turnaround time is about 6 h. • The array is relatively cheap.

  24. Relevant publications • Yang et al. 2011. Journal of Clinical Microbiology. 49:797-801. • Lu et al. 2011. Journal of Clinical Microbiology. 49:2290–2292. • Hung et al. 2011. BMC Infectious Diseases. 11:91 • Lu et al. 2013. Diagnostic Microbiology and Infectious Disease. 75:337-341. • Huang et al. 2014. Journal of Microbiology, Immunology and Infection. doi: 10.1016/j.jmii.2014.02.001. • Huang et al., 2014. Clinical Microbiology and Infection. 20: O542–O549. • Chien et a.. 2015. PLOS ONE, DOI:10.1371/journal.pone.0125016.

  25. Special thanks • Wen-Chun Yen, Hsin-Hui Hunag • Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwan • Chia-Jung Chiang, Meng-Hsun Chen • Chest Hospital, Department of Health, Tainan, Taiwan • Ministry of Science and Technology, Taiwan

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