Molecular Diagnostic ofMycobacterium tuberculosis
for Primary Health Care
Ida ParwatiIndonesian Society of Clinical Pathologist and Laboratory Medicine
Department of Clinical Pathology Dr. Hasan Sadikin General HospitalFaculty of Medicine Universitas Padjadjaran
B A N D U N G
20170408 Molecular diagnostic TB_HKKI
Outline
• Epidemiology: Global TB report 2016• WHO recommended technology of TB Diagnostic• Mycobacterium tuberculosis genome• Mekanisme Resistensi• Pemeriksaan Laboratorium TB dan MDRTB dan
permasalahannya
20170408 Molecular diagnostic TB_HKKI
20170408 Molecular diagnostic TB_HKKI
TB Diagnosis: WHO Recommended Technology
20170408 Molecular diagnostic TB_HKKI WHO,2016
20170408 Molecular diagnostic TB_HKKIWHO,2016
Indonesia: Endorsement Pemerintah untuk Tes Cepat Molekuler TCM)
20170408 Molecular diagnostic TB_HKKI
LAMPIRANPERATURAN MENTERI KESEHATAN REPUBLIK INDONESIANOMOR 67 TAHUN 2016TENTANGPENANGGULANGAN TUBERKULOSIS
20170225 SIMPLE TO AD TB DIAGNOSTIC_MAKASSAR
‘Deciphering the biology ofMycobacterium tuberculosisfrom the complete genome
sequence’
Cole, Nature,95
20170408 Molecular diagnostic TB_HKKI
Farhat et al. Genomic analysis identifies targetsof convergent positive selection in drug-
resistant Mycobacterium tuberculosis.Nature Genetics 45, 1183-1189 (2013)
20170408 Molecular diagnostic TB_HKKI
EMB
INH
RIF
FQ
σ
H+
Acidification ofcytoplasma
HPOA
H+
H+
H+
POA -
HPOA POA -PZA
PZase
arabinan + D-arabinose
arabinogalactan
Detoxification oforganic peroxides
alkylhydroperoxidase
EnoylACP
NADH
activated INHisoNAD
competitiekatalase
celwallsynthesis
celwalsynthesis
katGembBembAembC
pncA
rpoB “hotspot”
arabinosyldonor
ahpC
embCABeiwitten
Interuption ofmembrane function
gyrase
mRNARNA
polymeraseα β'β
gyrA
σ
inhA
accumulation ofmycolinic acids
20170408 Molecular diagnostic TB_HKKI
R van Crevel,Parwati 2009
Xpert MTB/RIF: Test and Treattwo-hour detection of MTB and RIF resistance
Sensitive:• Hemi-nested PCR increases sensitivity• Highly sensitive for confirmation of both smear + and smear -samples• Robust sonication/mechanical DNA extraction procedure• Internal extraction control assures extraction performance:Spesific:• Uses 3 specific primers and 5 unique molecular probes to ensure a high specificity• Targets the rpoB gene, for identifying mutations associated with rifampicin resistance• No cross reactions with many other bacterial/Mycobacteria species (NTM)
20170408 Molecular diagnostic TB_HKKI
Xpert MTB/RIF
Advantages
• Detect TB and rif resistance at the same time• Rapid (results in in less than two hours)• High sensitivity (88%) and high specificity
(99%) when compared to liquid culture insputum samples
• Low biosafety level requirements (comparedto culture) and similar to microscopy
• Minimal training of personnel• Can detect both pulmonary TB and EPTB• Same system can be used for other conditions
such as early infant HIV diagnosis and viralload monitoring (pending WHO approval)
Disadvantages
• Requires stable uninterruptable electricity• Operating temperature should not >30ᴏC &
cartridge must be stored at <28oC (in an air-conditioned room)
• Cartridges’ shelf life must be monitored or areprone to being wasted (MTB/RIF’s shelf life iscurrently 22 months; Ultra, if approved, willlikely have a shorter shelf life)
• Security measures must be put in place toavoid theft of laptop or desktop computer
• Cannot be used to monitor TB treatment• The instrument’s accuracy needs to be
checked (calibrated) every year
20170408 Molecular diagnostic TB_HKKI
Source: Implementing TB diagnostics. World Health Organization. 2015 Geneva: WHO policyframework. http://apps.who.int/iris/bitstream/10665/162712/1/9789241508612_eng.pdf?ua=1(Accessed 2016 July 28)
Kodon 507-511
Kodon 512-518
Kodon 518-523Kodon 523-529
Kodon 529-533
HOT SPOT region>95% resistensi Rif akibat mutasi di hot spot
DETECTION TARGET:rpoB gene 81 bp Rif Resistance Determining Region
Molecular Beacon Technology• Molecular beacon is a short
segment of single-strandedDNA composed of:
• Target specific sequence
• Stem
• Fluorescent dye
• Quencher
20170408 Molecular diagnostic TB_HKKI
Molecular Beacon
Target
Fluorescence No Fluorescence
No Target = No Fluorescence
Molecular Beacon Technology
20170408 Molecular diagnostic TB_HKKI
20170408 Molecular diagnostic TB_HKKI
20170408 Molecular diagnostic TB_HKKI
Xpert MTB/RIF assay & GeneXpert instrument
20170408 Molecular diagnostic TB_HKKI
Xpert MTB/RIF assay & GeneXpert instrument
20170408 Molecular diagnostic TB_HKKI
Xpert MTB/RIF assay & GeneXpert instrument
20170408 Molecular diagnostic TB_HKKI
Detection of mutations that cause rifampin resistance in M.tuberculosis by the amplification of the rpoB core region in the
presence of five differently colored molecular beacons.
El-Hajj H H et al. J. Clin. Microbiol. 2001;39:4131-4137
20170408 Molecular diagnostic TB_HKKI
2017 launch of new TB test Ultra backed by WHO recommendationXpert MTB/RIF Ultra can better diagnose TB in children and peopleliving with HIV
“The Xpert MTB/RIF Ultra assay was redesigned to boost analytical sensitivity morethan ten-fold and to improve reliability of detecting mutations associated withrifampin resistance. The latter was accomplished by introducing a new and powerfulfeature, automated post-PCR melt curve analysis, to the GeneXpert System,” saidDavid H. Persing, MD, Ph.D., Cepheid’s Chief Medical and Technology Officer.”Ultra performance approaches that of liquid culture, but is faster and easier to use atthe point of care,” said Dr David Alland, Professor of Medicine and Director of theCenter of Emerging Pathogens, Rutgers New Jersey Medical School, whose researchlaboratory contributed to the development of the test.
20170408 Molecular diagnostic TB_HKKI
Xpert MTB/RIF Ultra: A New Near-Patient TB TestWith Sensitivity Equal to Culture
• Author(s):David Alland1, Mazhgan Rowneki1, Laura Smith1, Jamie Ryan2, Mitchell Chancellor2, Ann Marie Simmons2, DavidPersing2, Robert Kwiatkowski2, Martin Jones2, Soumitesh Chakravorty11 Medicine, Rutgers New Jersey Medical School, Newark, NJ, United States. 2 Cepheid, Sunnyvale, CA, UnitedStates.
20170408 Molecular diagnostic TB_HKKI
Conclusions. The new Ultra assay is much more sensitive than Xpert, and is likely tobe as sensitive as liquid TB culture. Ultra detects RIF-R as efficiently as Xpert; but thespecificity of Ultra RIF-R is likely to be higher due to improvements in assay design.The Ultra assay should significantly increase TB detection in smear-negative patientsand provide more reliable RIF-R detection.
Positive result - different TB diagnostic tools%
pos
itive
0
10
20
30
40
50
60
70
80
ZN Ogawa MB/BacT PCR IS6110
HIV pos(n=35)
HIV neg(n=166)
72
1710
26
40 4046
51
Lydia, 2011
20170408 Molecular diagnostic TB_HKKI
20170408 Molecular diagnostic TB_HKKIECCMID London 1-3 April 2012
20170408 Molecular diagnostic TB_HKKI
1 pg1 pg
100 fg100 fg
gyrBgyrB
gyrBgyrB
M.univM.univ
M.tbM.tb
- - + + + + + +
+ + + + + - - -
+ + + + - - - -
Lisdawaty, 2011, disertasi20170408 Molecular diagnostic TB_HKKI
Conjugate ctrl
rpoB wild-type, 5segments
4 rpoB mutations
rpoB universal ctrl
MTBDR by HAIN Lifescience LiPA RIF.TB byINNOGENETICS
katG universal ctrl
katG wild-type
2 katG mutations
Universal ctrlMTBC
516
526531
315
marker line
MTBC
More probes are added in MTBDRsl to detected 2nd-line drug R-mutations.20170408 Molecular diagnostic TB_HKKI
20170408 Molecular diagnostic TB_HKKI
GenoType MTBDRsl Ver2.0
20170408 Molecular diagnostic TB_HKKI
20170408 Molecular diagnostic TB_HKKI
Conclusion
• Simple, rapid, accurate, affordable, TB/MDR-TB tests is needed:to break the TB transmission chainrapid initiate TB/MD-TB treatment
20170408 Molecular diagnostic TB_HKKI
•terimakasih
20170408 Molecular diagnostic TB_HKKI