molecular diagnosis of drug resistant tuberculosis by a dna array tsung chain chang ( 張長泉 )...

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

Tuberculosis (TB)

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

Mycobacterium tuberculosis complex (MTBC)

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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.

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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.

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Global (2013) : 3.5%

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Global (2013) : 20.5%


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Laboratory diagnosis

Clinical specimensClinical specimens

MGITMGIT

LJLJ7H117H11

Drug susceptibility testDrug susceptibility test

Tim

eline (w

eeks)

01 ~

2

58

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Mycobacterial Growth Indicator Tube system (MGIT, BD)

Fluorescent on the bottom is sensitive to the presence of oxygendissolved in the broth. As the oxygen is used by microorganisms,the fluorescence can be detected.

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GenoType MTBDRplus (Hain Lifescience GmbH, Nehren, Germany)

• A line probe hybridization assay.

• Detection of rifampin and isoniazid resistance

• GenoType MTBDRsl assay

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Xpert MTB/RIF (Cepheid)

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

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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

DNA extraction >> heminested PCRIncubator 15min

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• 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.

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• 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.

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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.

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FIND Diagnostics. FIND. Retrieved 6 April 2014.

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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.

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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.

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Drug susceptibility testing (agar proportion method)

control INH

0.2 μg/ml

RIF

1 μg/ml

EMB

7.5 μg/ml

Medium : Middlebrook 7H10 agar medium.RIF: rifampin; INH: isoniazid; EMB: ethambutol

×100% ResistantNo. of colonies on drug-containing mediumNo. of colonies on drug-free medium=

If 1% resistance

Results are reported at three weeks after inoculation.

CLSI M24-A, 2003.

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The limits of culture methods

Culture&

identification

Drug susceptibility

test(First-line)

Drug susceptibility

test(Second-line)

Agar proportional methodMolecular

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.

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Treatment of TBFirst-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.

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Development of an array

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

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546 clinical isolates (204 patients)

Discrepant analysis

Oligonucleotide array

Agar proportional method

Agar proportional method

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.

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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.

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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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molecular diagnosis of drug resistant tuberculosis by a dna array tsung chain chang ( 張長泉 )...

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