Molecular Diagnostic of Mycobacterium tuberculosis - hkki. · PDF filePENANGGULANGAN...

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



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)


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


Farhat et al. Genomic analysis identifies targetsof convergent positive selection in drug-

resistant Mycobacterium tuberculosis.Nature Genetics 45, 1183-1189 (2013)


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


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)


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


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


Molecular Beacon

Target

Fluorescence No Fluorescence

No Target = No Fluorescence

Molecular Beacon Technology




Xpert MTB/RIF assay & GeneXpert instrument






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


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.


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.


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_HKKIECCMID London 1-3 April 2012


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


GenoType MTBDRsl Ver2.0



Conclusion

• Simple, rapid, accurate, affordable, TB/MDR-TB tests is needed:to break the TB transmission chainrapid initiate TB/MD-TB treatment


•terimakasih


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