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

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

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.

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.

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.

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

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

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

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 http://www.finddiagnostics.org/media/press/ 090324.html

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(+)

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.

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.

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.

FIND Diagnostics. FIND. Retrieved 6 April 2014.

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. 2011. J. Clin. Microbiol. 49:797-801.

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.

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.

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.

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.

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

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

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.

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.

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