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Istituto Nazionale per le malattie Infettive L. Spallanzani Roma, Italy Immunodiagnosis of TB Delia Goletti Borstel, May 28 th, 2010.

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Presentation on theme: "Istituto Nazionale per le malattie Infettive L. Spallanzani Roma, Italy Immunodiagnosis of TB Delia Goletti Borstel, May 28 th, 2010."— Presentation transcript:

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2 Istituto Nazionale per le malattie Infettive L. Spallanzani Roma, Italy Immunodiagnosis of TB Delia Goletti Borstel, May 28 th, 2010

3 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests

4 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests

5 Tuberculosis transmission and progression to active disease from latent infection Small PM et al, N Engl J Med, 2001

6 The challenge of detecting M. tuberculosis infection Active disease Often difficult to isolate: even with good microbiological facilities, the bacillus is recovered in only 60% of cases Latent infection M. tuberculosis cannot be cultured from latently infected individuals: no gold standard

7 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests

8 Tuberculin skin test-1 Reagent: Purified protein derivative (PPD) commonly shared among different Mycobacteria (M.tuberculosis, BCG and atypical mycobacteria) Variability: Reproducibility in giving the test Subjectivity in reading the test Logistics Repeat visit needed 3 days before result

9 Positive TST M. tuberculosis Active TB disease Latent TB infection NTMExposure to environmental mycobacteria BCG-vaccination Tuberculin skin test (TST)-2 TST does not distinguish among all these different clinical situations

10 Tuberculin skin test-3 False Negatives skin reaction is a very crude measure: Small responses not picked up (real problem in immunosuppressed patients) 10-25% negative results in active disease (only 75-90% sensitivity, worse in immunosuppressed)

11 Need of… Standardized test (laboratory test) M. tuberculosis-specific reagents Possibility to discriminate between the different stages of tuberculosis

12 Need of… Standardized test (laboratory test) M. tuberculosis-specific reagents Possibility to discriminate between the different stages of tuberculosis

13 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests

14 QuantiFERON ® -TB Nil Tuberculin PPD M. Avium PPD Mitogen

15 QuantiFERON ® -TB Test Method (in ) Heparinised whole blood Avian PPD Tuberculin PPD Mitogen Control Transfer undiluted whole blood into wells of a culture plate and add antigens Culture overnight at 37 o C TB infected individuals respond by secreting IFN- Harvest Plasma from above settled cells and incubate 60 min in Sandwich ELISA Wash, add Substrate, incubate 30 min then stop reaction TMB COLOR IFN- IU/ml OD 450nm Standard Curve Measure OD and determine IFN- levels Stage 1: Whole Blood Culture Nil Control Stage 2: IFN- ELISA

16 16 QuantiFERON ® -TB Test ResultNilTuberculin PPD M. avium PPD Mitogen Indeterminate–––– Negative–––+++ M. tuberculosis, atypical mycobacteria infection different from M. avium and BCG vaccination –++++ M. Avium infection –++++

17 Need of… Standardized test (laboratory test) M. tuberculosis-specific reagents Possibility to discriminate between the different stages of tuberculosis

18 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests

19 Species specificities of ESAT-6 and CFP-10 Environmental strains Antigens ESATCFP M abcessus-- M avium-- M branderi-- M celatum-- M chelonae-- M fortuitum-- M gordonii-- M intracellulare-- M kansasii++ M malmoense-- M marinum++ M oenavense-- M scrofulaceum-- M smegmatis-- M szulgai++ M terrae-- M vaccae-- M xenopi-- Tuberculosis complex Antigens ESATCFP M tuberculosis++ M africanum++ M bovis++ BCG substrain gothenburg-- moreau-- tice-- tokyo-- danish-- glaxo-- montreal-- pasteur--

20 Agenda Problems in the diagnosis of TB TST QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests

21 T-SPOT TB Lalvani et al, JID 2001 ESAT-6: 17 peptides CFP-10: 18 peptides

22 How the T-SPOT TB technology works Collect white cells using BD CPT tube or Ficoll extraction. Add white cells and TB antigens to wells. T cells release interferon-. Interferon- captured by antibodies. Incubate, wash and add conjugated second antibody to interferon- Add substrate and counT-SPOTs by eye or use reader. Each spot is an individual T cell that has released interferon-

23 T-SPOT TB

24 . T-SPOT TB is a patented method to detect pathogen-specific T cells. A simplified variant of the ex vivo elispot method developed by Dr. Ajit Lalvani Complete system - kit + instrumentation Validated and produced to international quality standards (ISO13485:2003, GMP) Standardized Quality-controlled CE marked for in vitro diagnostic use

25 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests

26 QuantiFERON ® -TB Gold In tube : peptides used Mori et al, AJRCCM 2004 ESAT-6: 7 peptides CFP-10: 6 peptides TB7.7: 1 peptide

27 QuantiFERON ® -TB Gold In tube: methods Collect peripheral venous blood (4 or 8 ml) Harvest plasma Keep the plasma harvested at: - 4 C for 15 days - 20 C for 2 months ELISA Incubate overnight at 37˚C Centrifuge for 5 minutes SOFTWARE for DATA ANALYSIS

28 Stage 1: Culture overnight at 37 o C M.tuberculosis-infected individuals respond by secreting IFN- Harvest Plasma from above settled cells and incubate 120 min in Sandwich ELISA Wash, add Substrate, incubate 30 min then stop reaction TMBCOLOR Measure OD and determine IFN- levels Stage 2: IFN- ELISA IFN- IU/ml OD 450nm Standard Curve QuantiFERON ® -TB Gold In tube

29 Comparison TST vs IGRA TSTRD1 IGRA ELISPOT (e.g. T-SPOT TB) ELISA ( e.g. QuantiFERON-TB Gold IT ) Antigens PPDPeptides from CFP-10, ESAT-6 Peptides from CFP-10, ESAT-6 and TB7.7 Tests substrate SkinPBMCWhole Blood Time required for the results 72 h24 h Cells involved Neutrophils, CD4, CD8 that transmigrate out of capillaries into the skin. Treg (CD4 + CD25 high FoxP3 + ). CD4 T cells in vitro Cytokines involved IFN-, TNF-, TNF- IFN- Modified from Mack et al, ERJ 2009

30 Comparison TST vs IGRA TSTRD1 IGRA ELISPOT (e.g. T-SPOT TB) ELISA ( e.g. QuantiFERON-TB Gold IT ) Read-out Measure of diameter of dermal induration Enumeration of IFN- spots Measure of optical density values of IFN- production Outcomes measure Level of induration Number of IFN- producing T cells Plasma concentration of IFN- produced by T cells Read-out units mm IFN- spot forming cells IU/ml Modified from Mack et al, ERJ 2009

31 Comparison TST vs IGRA TSTRD1 IGRA ELISPOT (e.g. T-SPOT TB) ELISA ( e.g. QuantiFERON-TB Gold IT) Internal control noyes Technical expertise required Medium high Low medium Cost of reader machine -Medium highLow medium Cost of the assay 2-3 euros euros? Modified from Mack et al, ERJ 2009

32 Accuracy TST T-SPOT TB QuantiFERON-TB Gold

33 TST sensitivity SENSITIVITY TST 77% Pai et al, Annals 2008

34 TST specificity SPECIFICITY TST 97% SPECIFICITY TST 59% in those BCG-vaccinated Pai et al, Annals 2008

35 Sensitivity T-SPOT TB vs QuantiFERON TB- Gold In tube Pai et al, Annals 2008 SENSITIVITY T SPOT TB 90% SENSITIVITY QFT-IT 70%

36 Specificity T-SPOT TB vs QuantiFERON TB- Gold In tube Pai et al, Annals 2008 SPECIFICITY T SPOT TB 93% SPECIFICITY QFT-IT 96%

37 Positive RD1-IGRA Positive M. tuberculosis infection/disease NTM BCG-vaccination RD1-IGRA Positive RD1-IGRA do not distinguish active TB disease and LTBI Active TB disease Latent TB infection

38 Vulnerable populations Children Immuno-suppressed for: HIV Autoimmune disease

39 Comparison of TST/IGRAs in children with active TB SourcePatient number TST + % T-SPOT TB + % QTF-G + % To note Liebeschuetz et al, Lancet NA TB microbiologically diagnosed Kampmann et al, ERJ TB microbiologically diagnosed Hermann JL et al, Plos NA78 TB microbiologically diagnosed in 48% Nicol et al, Pediatrics NA TB microbiologically diagnosed Connell et al, Plos TB clinically diagnosed

40 Comparison of TST/IGRAs in patients HIV + with active TB microbiologically confirmed SourcePatient number TST + % T-SPOT TB + % QTF-G + % RD1 proteins ELISPOT + % To note Liebeschuetz et al, Lancet nd T cell anergy no reported, CD4 nd Vincenti et al, Clin Exp Imm ndT cell anergy in 20%, CD4 median 179 Raby et al, Plos ONE nd75ndT cell anergy in 17%, CD4 median 212 Aabye et al, Plos ONE nd 83ndT cell anergy in 22%, CD4 median 179 Rangaka et al, CID nd 90T cell anergy no reported, No commercial test, CD4 median 167

41 Comparison of TST/IGRAs in patients with active TB microbiologically confirmed SourcePatient number TST + % T-SPOT TB + % QTF-G + % RD1 proteins ELISPOT + % To note Liebeschuetz et al, Lancet nd T cell anergy no reported, CD4 nd Vincenti et al, Clin Exp Imm ndT cell anergy in 20%, CD4 median 179 Raby et al, Plos ONE nd75 63 ndT cell anergy in 17%, CD4 median 212 Aabye et al, Plos ONE nd ndT cell anergy in 14%, CD4 median 179 Rangaka et al, CID nd 90T cell anergy no reported, No commercial test, CD4 median 167

42 Proportion of in vitro anergic responses to IGRAs in HIV + patients Brock, Resp Res 2007 Vincenti, Clin Exp Imm 2007 Luetkem eyer, AJRCCM 2007 Clark, Clin Exp Imm 2007 Karam, Plos ONE 2008 Rabi, Plos ONE 2008 Test QFTELISPOT home-made QFT ELISPOT home-made QFT N. Paz CD4 per l <1004 (24%)12 (57%)5 (16%)4 (6%)6 (16%)6 (46%) (3%)4 (19%)4 (3.6%) 1 (NA) 12 (31%)3 (15%) (8%)3 (14%)10 (26)3 (13%) >30010 (2%)2 (10%)6 (3.9%)8 (21%)4 (8%)

43 Immunocompromised for immune suppressive therapy due to autoimmune diseases Bartalesi et al, ERJ 2009

44 Predictive value of IGRA: HIV-negative subjects Diel et al, AJRCCM 2009

45 Predictive value of IGRA: HIV + subjects Aichelbuurg et al, CID 2009

46 Predictive value of IGRA: HIV-negative subjects Kik et al, ERJ 2009

47 Need of… Standardized test (laboratory test) M. tuberculosis-specific reagents Possibility to discriminate between the different stages of tuberculosis

48 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests IGRA based on RD1 selected peptides or on antigens of latency IP-10 detection IGRA at the site of TB disease Ratio IL-2/IFN-

49 Why is it important to distinguish between latent infection and active TB disease? To provide a correct diagnosis: Active TB disease: Organ destruction and/or death Spread of infection in the community Latent infection To provide a correct and efficacious therapy: Active TB disease: 2 months therapy with 4 drugs and the 2 months therapy with 2 drugs Latent TB infection: 6 months therapy with one drug To save human and economic costs avoiding complex evaluations (i.e. clinical, radiological and surgery procedures). Ex: extra-pulmonary TB

50 Our approach: use of peptides from ESAT-6 and CFP-10 selected by computational analysis PeptidePosition sequenceDR-serological specificities covered 1- ESAT , 3, 4, 8, 11(5), 13(6), 52, ESAT , 8, 11(5), 13(6), 15(2), CFP , 5, 11(5), CFP , 3, 4, 7, 8, 11(5),13(6), 15(2), CFP , 4, 7, 11(5), 12(5), 13(6), 15 (2) Peptides selected by computational analysis that cover more than 90% of the HLA class II specificities

51 IFN- response to RD1 selected peptides is associated to active TB Vincenti et al, Mol Med 2003

52 In patients with active TB the response to RD1 selected peptides decreases after efficacious treatment Carrara et al, CID 2004

53 Sensitivity, specificity and diagnostic odds ratio of the different assays for the immune diagnosis of TB AssaySensitivity % Specificity % Diagnostic odds ratio RD1 ELISPOT assays Selected peptides Intact proteins RD1 commercially available assays T-SPOT TB QFT Gold83597 Goletti et al, CMI 2006

54 TBNET report Goletti et al, PLoS ONE 2008

55 In vitro IFN- response to Rv2628 antigen of latency is associated with remote LTBI Goletti et al, ERJ 2010

56 LTBI vs Active TB: HBHA Berlin, October 4th, 2008 From Hougardy et al, 2007

57 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests IGRA based on RD1 selected peptides or on antigens of latency IGRA at the site of TB disease IP-10 detection Ratio IL-2/IFN-

58 IGRA at the site of TB disease: BAL vs blood From Jafari, AJRCCM 2009

59 IGRA at the site of TB disease: Pleural fluid vs blood Berlin, October 4th, 2008 From Losi et al, ERJ 2007 PLEURAL CELLSPBMC

60 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests IGRA based on RD1 selected peptides or on antigens of latency IGRA at the site of TB disease IP-10 detection Ratio IL-2/IFN-

61 IP-10 is induced by ESAT-6, CFP10 e TB7.7 in whole blood from patients with TB disease QFT-Gold, detection of: IP-10 (Ruwald, 2007): Significant higher in patients with active disease IP-10 detectable in patients with active TB scored negative by IFN- detection of the QFT-Gold

62 Detection of IP-10 in the plasma harvested from QuantiFERON-TB Gold In-tube From Ruwald et al, modified Microbes Infection 2007

63 IP-10 and MCP-2 in the plasma harvested from QuantiFERON-TB Gold In-tube are associated with active TB From Ruwald et al, ERJ 2008

64 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests IGRA based on RD1 selected peptides or on antigens of latency IGRA at the site of TB disease IP-10 detection Ratio IL-2/IFN-

65 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests IGRA based on RD1 selected peptides or on antigens of latency IP-10 detection IGRA at the site of TB disease Ratio IL-2/IFN-

66 Dynamic relationship between IFN- and IL-2 profile of M. tuberculosis-specific T cells and antigen load From Millington, J Immunol 2007, modified

67 Agenda Problems in the diagnosis of TB TST IGRA QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests IGRA based on RD1 selected peptides or on antigens of latency IP-10 detection IGRA at the site of TB disease Ratio IL-2/IFN-

68 And thank you to:


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