1. 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
7th Asia-Pacific Biotech Congress
July 13-15, 2015, Beijing, China

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

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-resistant TB)
resistant to at least rifampin and isoniazid.
XDR-TB (extensively drug-resistant TB)
MDR-TB with resistance to a fluoroquinolone
and either capreomycin, amikacin, or kanamycin.

4. introduction
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. Extensively drug-resistant TB (XDR-TB) has been reported by 100 countries. On average, an estimated 9.0% (95% CI: 6.5–11.5%) of people with MDR-TB have XDR-TB.
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. Drug susceptibility test
Laboratory diagnosis
Clinical specimens
Timeline (weeks)
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 90 minutes
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11. DNA extraction >> heminested PCR
Incubator 15min
DNA extraction >> heminested PCR
B. globigii DNA 會跟著PCR被放大，可做為是否有M.tuberculosis/RIF resistance及M. tuberculosis的半定量評估。當Assay測得M. tuberculosis為Negative，則B. globigii也會是Negative.
偵測rpoB gene mutation：利用corresponding probes水解rpoB-specific molecular beacons，而mutation的gene不會被水解，所以當rpoB-specific molecular beacon五個至少兩個被測得時，就是為rpoB gene mutation(+)

12. Xpert MTB/RIF (Cepheid)
Results are obtained from unprocessed sputum samples in 90 minutes
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 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 Cochrane Database of Systematic Reviews 2013: DOI: / CD pub2.
Boehme et al. N Engl J Med. 2010; 363:

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

16. Evaluation of the Cobas TaqMan MTB Test for Direct Detection of MTBC in 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 J. Clin. Microbiol. 49:

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 J. Clin. Microbiol. 49:

18. Drug susceptibility testing
(agar proportion method)
×100
% Resistant
No. of colonies on drug-containing medium
No. of colonies on drug-free medium =
If 1%  resistance
Results are reported at three weeks after inoculation.
control
INH
0.2 μg/ml
Inoculums :
1. colonies from the surface of the agar medium
2. 1 McFarland
3. dilutions 10-2,10-4
Incubate at 37±1 ℃, 5 ~10 % CO2
RIF
1 μg/ml
EMB
7.5 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
(First-line)
Drug susceptibility test
(Second-line)
Culture
&
identification
Molecular method
Agar proportional method
Many XDR-TB cases are not diagnosed since testing for resistance to 2nd-line drugs is not routinely performed.
Blaschitz et al., 2011
Accurate diagnosis and early initiation of treatment are important to reduce transmission and hinder the emergence of drug-resistant TB Ajbani et al., 2011.
Conventional methods to detect drug resistant TB are laborious and time-consuming.

20. Treatment of TB First-line drugs
Isoniazid (INH); Rifampin (RIF); Pyrazinamide (PZA); Ethambutol (EMB); Streptomycin (SM).
Second-line drugs
Capreomycin; Ofloxacin; Levofloxacin; Moxifloxacin; Rifabutin; Kanamycin; Amikacin; Prothionamide; Para-aminosalicylate; Cycloserine; Ethionamide.
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)
Oligonucleotide array
Agar proportional method
Discrepant analysis

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 Journal of Clinical Microbiology. 49:
Lu et al Journal of Clinical Microbiology. 49:2290–2292.
Hung et al BMC Infectious Diseases. 11:91
Lu et al Diagnostic Microbiology and Infectious Disease. 75:
Huang et al Journal of Microbiology, Immunology and Infection. doi: /j.jmii
Huang et al., Clinical Microbiology and Infection. 20: O542–O549.
Chien et a PLOS ONE, DOI: /journal.pone

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