Molecular Diagnostic of Mycobacterium tuberculosis - HKKI [PDF]

Indonesia: Endorsement Pemerintah untuk Tes Cepat Molekuler TCM). 20170408 Molecular diagnostic TB_HKKI. LAMPIRAN. PERAT

5 downloads 4 Views 1MB Size

Recommend Stories


Molecular Identification of Mycobacterium Tuberculosis in the [PDF]
Osteological Paradox. Wood's groundbreaking 1992 “The Osteological Paradox: Problems of Inferring. Prehistoric Health from Skeletal Samples” questions the utility of inferring the health of populations from skeletal remains. If all of the people

Mycobacterium tuberculosis
Be grateful for whoever comes, because each has been sent as a guide from beyond. Rumi

Mycobacterium tuberculosis
Just as there is no loss of basic energy in the universe, so no thought or action is without its effects,

Mycobacterium tuberculosis
Knock, And He'll open the door. Vanish, And He'll make you shine like the sun. Fall, And He'll raise

Mycobacterium tuberculosis
When you do things from your soul, you feel a river moving in you, a joy. Rumi

Mycobacterium tuberculosis
Don't be satisfied with stories, how things have gone with others. Unfold your own myth. Rumi

Mycobacterium tuberculosis
What you seek is seeking you. Rumi

Mycobacterium tuberculosis
The best time to plant a tree was 20 years ago. The second best time is now. Chinese Proverb

Mycobacterium tuberculosis
Your big opportunity may be right where you are now. Napoleon Hill

Mycobacterium tuberculosis
What you seek is seeking you. Rumi

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

Smile Life

When life gives you a hundred reasons to cry, show life that you have a thousand reasons to smile

Get in touch

© Copyright 2015 - 2024 PDFFOX.COM - All rights reserved.