Idea Transcript
Molecular Diagnostic of
Mycobacterium tuberculosis for Primary Health Care Ida Parwati Indonesian Society of Clinical Pathologist and Laboratory Medicine Department of Clinical Pathology Dr. Hasan Sadikin General Hospital Faculty of Medicine Universitas Padjadjaran BANDUNG 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_HKKI
WHO,2016
Indonesia: Endorsement Pemerintah untuk Tes Cepat Molekuler TCM) LAMPIRAN PERATURAN MENTERI KESEHATAN REPUBLIK INDONESIA NOMOR 67 TAHUN 2016TENTANG PENANGGULANGAN TUBERKULOSIS
20170408 Molecular diagnostic TB_HKKI
20170225 SIMPLE TO AD TB DIAGNOSTIC_MAKASSAR
‘Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence’
Cole, Nature,95
20170408 Molecular diagnostic TB_HKKI
Farhat et al. Genomic analysis identifies targets of convergent positive selection in drugresistant Mycobacterium tuberculosis. 20170408 Molecular diagnostic TB_HKKI
Nature Genetics 45, 1183-1189 (2013)
H+ H+ HPOA
EMB
POA
-
POA
-
H+ celwalsynthesis
arabinosyl donor
arabinan + Darabinose embC
FQ
PZase
arabinogalactan
embCAB eiwitten
HPOA
accumulation of mycolinic acids embA
embB
gyrase
H+ Acidification of cytoplasma
Interuption of membrane function
inhA
gyrA
PZA
pncA
katG
activated INH isoNAD
katalase
INH
competitie NADH mRNA
RIF rpoB “hotspot”
RNA polymerase α β β' σ
Detoxification of organic peroxides Enoyl ACP
celwallsynthesis 20170408 Molecular diagnostic TB_HKKI
ahpC alkyl hydroperoxidase
R van Crevel, Parwati 2009
Xpert MTB/RIF: Test and Treat two-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
Disadvantages
• 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 in sputum samples • Low biosafety level requirements (compared to 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 viral load monitoring (pending WHO approval)
• Requires stable uninterruptable electricity ᴏ • Operating temperature should not >30 C & cartridge must be stored at 95% resistensi Rif akibat mutasi di hot spot
Kodon 523-529 Kodon 507-511
Kodon 518-523
Kodon 512-518
Kodon 529-533
Molecular Beacon Technology • Molecular beacon is a short segment of single-stranded DNA composed of: • Target specific sequence • Stem • Fluorescent dye • Quencher
20170408 Molecular diagnostic TB_HKKI
Molecular Beacon Technology Molecular Beacon
Target
Fluorescence
No Fluorescence
No Target = No Fluorescence 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 recommendation Xpert MTB/RIF Ultra can better diagnose TB in children and people living with HIV “The Xpert MTB/RIF Ultra assay was redesigned to boost analytical sensitivity more than ten-fold and to improve reliability of detecting mutations associated with rifampin resistance. The latter was accomplished by introducing a new and powerful feature, automated post-PCR melt curve analysis, to the GeneXpert System,” said David 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 at the point of care,” said Dr David Alland, Professor of Medicine and Director of the Center of Emerging Pathogens, Rutgers New Jersey Medical School, whose research laboratory contributed to the development of the test. 20170408 Molecular diagnostic TB_HKKI
Xpert MTB/RIF Ultra: A New Near-Patient TB Test With Sensitivity Equal to Culture • Author(s): David Alland1, Mazhgan Rowneki1, Laura Smith1, Jamie Ryan2, Mitchell Chancellor2, Ann Marie Simmons2, David Persing2, Robert Kwiatkowski2, Martin Jones2, Soumitesh Chakravorty1 1 Medicine, Rutgers New Jersey Medical School, Newark, NJ, United States. 2 Cepheid, Sunnyvale, CA, United States.
Conclusions. The new Ultra assay is much more sensitive than Xpert, and is likely to be as sensitive as liquid TB culture. Ultra detects RIF-R as efficiently as Xpert; but the specificity 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 patients and provide more reliable RIF-R detection. 20170408 Molecular diagnostic TB_HKKI
Positive result - different TB diagnostic tools 80
72
% positive
70 60 51
50
46 40
40 30 20
26
HIV neg (n=166)
17 10
10 0
HIV pos (n=35)
40
ZN
Ogawa
MB/BacT
PCR IS6110
Lydia, 2011 20170408 Molecular diagnostic TB_HKKI
20170408 Molecular diagnostic TB_HKKI
ECCMID London 1-3 April 2012
20170408 Molecular diagnostic TB_HKKI
100 fg
gyrB
+ + + + + -
gyrB
+ + + +
-
-
-
-
M.univ
-
-
M.tb
1 pg
20170408 Molecular diagnostic TB_HKKI- - + + + + + + Lisdawaty, 2011, disertasi
MTBDR by HAIN Lifescience Conjugate ctrl MTBC
Universal ctrl
LiPA RIF.TB by INNOGENETICS marker line
MTBC
rpoB universal ctrl rpoB wild-type, 5 segments 516 526 531
4 rpoB mutations katG universal ctrl katG wild-type
315
2 katG mutations
20170408 Molecular diagnostic TB_HKKI More probes are added in MTBDRsl to detected 2nd-line drug R-mutations.
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 chain rapid initiate TB/MD-TB treatment
20170408 Molecular diagnostic TB_HKKI
•terimakasih 20170408 Molecular diagnostic TB_HKKI