Identifying TB co-infection : new approaches?

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1 Identifying TB co-infection : new approaches? Charoen Chuchottaworn MD. Senior Medical Advisor, Central Chest Institute of Thailand, Department of Medical Services, MoPH

2 Primary tuberculosis Natural history Chemotherapy 90% 10% 98% 1-3% Nature Rev Immune 2011 doi: /nri2960

3 Nature Rev 2016;2:1-23

4 Diagnosis of Latent Tuberculosis Infection Current standard tests - Tuberculin skin test (TST) - Interferon-γ release assays (IGRA) - QuantiFERON Gold-in-Tube (QTF-GIT) - T.SPOT-TB New tests - QuantiFERON TB-Gold Plus - Gene expression pattern - Cytokines response pattern - T cell activation markers

5 Diagnosis of Tuberculosis Clinical signs and symptoms X-ray findings Demonstration of mycobacteria: smear and culture Demonstration of mycobacterial DNA/RNA Demonstration of mycobacterial antigens or antibodies Demonstration of host responses Histopathology

6 Moving from smear and staining to molecular test WHOrecommended rapid diagnostic - Smear and staining is not sensitive ( sensitivity 30-60% ) - Smear positive need 30,000 bacilli/ml. - Molecular test is more sensitive ( sensitivity 80-90% ) - WHO-recommended rapid diagnostic - WRD = Xpert MTB/RIF, Loopamp MTBC detection kit.

7 Evaluation of 5 novel proteins biomarkers for the diagnosis of pulmonary and extra-pulmonary tuberculosis : preliminary result

8 Evaluation of 5 novel proteins biomarkers for the diagnosis of pulmonary and extra-pulmonary tuberculosis : preliminary result

9 Evaluation of 5 novel proteins biomarkers for the diagnosis of pulmonary and extra-pulmonary tuberculosis : preliminary result

10 PNAS 2017;114:

11 Diagnosis of Latent Tuberculosis Infection Current standard tests - Tuberculin skin test (TST) - Interferon-γ release assays (IGRA) - QuantiFERON Gold-in-Tube (QTF-GIT) - T.SPOT-TB New tests - QuantiFERON TB-Gold Plus - Gene expression pattern - Cytokines response pattern - T cell activation markers

12 Comparing interferon gamma assays and tuberculin skin test for identifying latent tuberculosis infection that progress to active tuberculosis systematic review and meta-analysis BMC Infect Dis 2017;

13 New Generation of IGRA Reasons of including CD8+ T cells stimulations are - CD8+ T cells can produce Inferferon-γ without CD4+ T cells - Response of CD8+ T cells correlates well with bacterial loads - Response of CD8+ T cells was decreased after 24 weeks of Rx Antigens used in both tests (QTF-GIT and QFTplus) QTF-GIT QTF plus one tube tube 1 (TB1) tube 2(TB2) ESAT6, CFP10, 7.7 Long chain of Short chain of ESAT6, CFP10 ESAT6, CFP10 nil tube nil tube mitogen tube mitogen tube

14 Evaluation of QuantiFERON-TB Gold Plus for Detection of Mycobacterium tuberculosis infection in Japan

15 Evaluation of QuantiFERON-TB Gold Plus for Detection of Mycobacterium tuberculosis infection in Japan

16 Eur Respir J 2016;48:

17 Eur Respir J 2016;48:

18 Eur Respir J 2016;48:

19 ESAT-6 CFP-10 Am J Respir Crit Care Med 2015;192:

20 PPD TB : infected vs uninfected = IFN-γ, IP-10,TNF-α, IL2 LTBI vs Disease = TNF-α, IL-1ra, IL-10 Am J Respir Crit Care Med 2015;192:

21

22

23 - Measure CD27 T- lymphocyte in Blood - Active TB had low CD27 T- lymphocyte in blood Lancet Inf Dis 2014;14:931-38

24 Gene Expression Assay Venepuncture of whole blood Convert to cdna Cell lysis and stabilize RNA with Extract RNA Prepare PCR reaction mixture and us cdna as template Generate nano-droplets ( approximately droplets) in PCR mixture (1 droplet = 1 nano-litre = 1 primer-pairs) PCR Quantitatify nano-droplets Demonstrate as Ratio to control house-keeping gene

25 Gene expression Quantitative PCR - Create nano-droplets - PCR in nano-droplets - Count positive and negative nanodroplets

26 J Mol Med 2007;85:613-21

27 J Mol Med 2007;85:613-21

28 Set of 393 genes Nature 2010;466:973-9.

29 Nature 2010;466:973-9.

30 N Eng J Med 2014;370:

31 N Eng J Med 2014;370:

32 N Eng J Med 2014;370:

33 Gene Immun 2015;16:253-60

34 Gene Immun 2015;16:253-60

35

36

37 Tuberculosis 2016;100:25-31.

38 Tuberculosis 2016;100:25-31.

39

40 BMC Med 2017;15:67-83

41 Summary - IGRA is useful test to determine tuberculosis infection but not correlate with activity of disease and alternative test of cytokines releasing assay profile seems to be more correlate with disease activity. - Serologic tests do not have high enough sensitivity to use in clinical practice unless new specific antigens could be defined. - Gene expression profile is a promising test to differentiate TB disease, latent tuberculosis infection progressing to disease and healthy patient. - Urine LAM antigen test is a useful routine test for identifying tuberculosis infection in HIV infected patients.

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