Transcript Journal of Clinical Microbiology

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
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Tuberculosis (TB)
Mycobacterium tuberculosis complex
(MTBC)
• TB is caused by Mycobacterium tuberculosis (MTB), a very
slow growing Gram-positive bacillus.
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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%
Global tuberculosis report 2014, WHO.
Taiwan Tuberculosis Control Report 2013, Taiwan CDC.
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Global (2013) : 20.5%
Global tuberculosis report 2014, WHO.
Taiwan Tuberculosis Control Report 2013, Taiwan CDC.
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Laboratory diagnosis
0
MGIT
1~2
LJ
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Drug susceptibility test
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7H11
Timeline (weeks)
Clinical specimens
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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.
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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)
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
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Incubator 15min
DNA extraction >> heminested PCR
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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.
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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.
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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 culturepositive 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)
No. of colonies on drug-containing medium
% Resistant = No. of colonies on drug-free medium
×100
If 1%  resistance
control
INH
0.2 μg/ml
RIF
Results are reported at three weeks
after inoculation.
EMB
1 μg/ml 7.5 μg/ml
Medium : Middlebrook 7H10 agar medium.
RIF: rifampin; INH: isoniazid; EMB: ethambutol
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 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 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.
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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
Discrepant
analysis
Agar proportional
method
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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. 75:337341.
• 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