Meeting INMI - Biomerieux • Diagnosis of Active & Latent TB August 25th, 2005 Istituto Nazionale per le malattie Infettive L. Spallanzani Roma, Italy Immunodiagnosis of TB Delia Goletti Borstel, May 28th, 2010 Je suis honorée d’être ici aujourd'hui pour présenter les résultats d’un nouveau test diagnostique de la tuberculose. Je ne parle pas bien le français scientifique, mais c’est avec plaisir que j’essayeraisde faire cette présentation dans votre merveilleuse langue. INMI, Goletti

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

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

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

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

Tuberculin skin test-1 Reagent: Variability: Logistics 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

Tuberculin skin test (TST)-2 Positive TST M. tuberculosis Active TB disease Latent TB infection NTM Exposure to environmental mycobacteria BCG-vaccination Le test de Man toux a été très utile. Toutefois la mantoux ne peut pas distinguer entre la maladie active, latente, la récente exposition a M. tuberculosis, ou a d’autre mycobactéries, ou a la vaccination au BCG. TST does not distinguish among all these different clinical situations

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)

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

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

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

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

QuantiFERON®-TB Test Method (in 2002...) Stage 1: Whole Blood Culture Nil Control Avian PPD Tuberculin PPD Mitogen Control Transfer undiluted whole blood into wells of a culture plate and add antigens Culture overnight at 37oC TB infected individuals respond by secreting IFN-g Heparinised whole blood Stage 2: IFN-g ELISA TMB COLOR IFN-g IU/ml OD 450nm Standard Curve Harvest Plasma from above settled cells and incubate 60 min in ‘Sandwich’ ELISA Wash, add Substrate, incubate 30 min then stop reaction Measure OD and determine IFN-g levels

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

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

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

Species specificities of ESAT-6 and CFP-10 Tuberculosis complex Antigens ESAT CFP M tuberculosis + M africanum M bovis BCG substrain   gothenburg - moreau tice tokyo danish glaxo montreal pasteur Environmental strains Antigens ESAT CFP 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 - -

Agenda Problems in the diagnosis of TB TST QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

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

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-g. Interferon-g captured by antibodies. Incubate, wash and add conjugated second antibody to interferon-g. Add substrate and counT-SPOTs by eye or use reader. Each spot is an individual T cell that has released interferon-g.

T-SPOT TB

T-SPOT TB 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

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests QuantiFERON-TB RD1-based assays: T-SPOT TB QuantiFERON-TB Gold New experimental tests Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

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

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

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

ELISA (e.g. QuantiFERON-TB Gold IT) Comparison TST vs IGRA TST RD1 IGRA ELISPOT (e.g. T-SPOT TB) ELISA (e.g. QuantiFERON-TB Gold IT) Antigens PPD Peptides from CFP-10, ESAT-6 Peptides from CFP-10, ESAT-6 and TB7.7 Tests’ substrate Skin PBMC Whole Blood Time required for the results 72 h 24 h 24h Cells involved Neutrophils, CD4, CD8 that transmigrate out of capillaries into the skin. Treg (CD4+CD25highFoxP3+). CD4 T cells in vitro Cytokines involved IFN-g, TNF-a, TNF-b IFN-g Modified from Mack et al, ERJ 2009

ELISA (e.g. QuantiFERON-TB Gold IT) Comparison TST vs IGRA TST RD1 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-g spots Measure of optical density values of IFN-g production Outcomes measure Level of induration Number of IFN-g producing T cells Plasma concentration of IFN-g produced by T cells Read-out units mm IFN-g spot forming cells IU/ml Modified from Mack et al, ERJ 2009

ELISA (e.g. QuantiFERON-TB Gold IT) Comparison TST vs IGRA TST RD1 IGRA ELISPOT (e.g. T-SPOT TB) ELISA (e.g. QuantiFERON-TB Gold IT) Internal control no yes Technical expertise required Medium high Low medium Cost of reader machine - Cost of the assay 2-3 euros 30-35 euros? Modified from Mack et al, ERJ 2009

Accuracy TST T-SPOT TB QuantiFERON-TB Gold Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

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

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

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

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

Positive M. tuberculosis infection/disease RD1-IGRA Positive RD1-IGRA Positive M. tuberculosis infection/disease NTM BCG-vaccination Active TB disease Latent TB infection Le test de Man toux a été très utile. Toutefois la mantoux ne peut pas distinguer entre la maladie active, latente, la récente exposition a M. tuberculosis, ou a d’autre mycobactéries, ou a la vaccination au BCG. Positive RD1-IGRA do not distinguish active TB disease and LTBI

Vulnerable populations Children Immuno-suppressed for: HIV Autoimmune disease

Comparison of TST/IGRAs in children with active TB Source Patient number TST+ % T-SPOT TB+ QTF-G+ To note Liebeschuetz et al, Lancet 2004 57 81 NA TB microbiologically diagnosed Kampmann et al, ERJ 2009 25 83 58 80 Hermann JL et al, Plos 2008 32 87 78 TB microbiologically diagnosed in 48% Nicol et al, Pediatrics 2009 10 50 Connell et al, Plos 2008 9 89 100 TB clinically diagnosed

Liebeschuetz et al, Lancet 2004 Comparison of TST/IGRAs in patients HIV+ with active TB microbiologically confirmed Source Patient number TST+ % T-SPOT TB+ QTF-G+ RD1 proteins ELISPOT+ % To note Liebeschuetz et al, Lancet 2004 57 35 81 nd T cell anergy no reported, CD4 nd Vincenti et al, Clin Exp Imm 2007 13 46 85 T cell anergy in 20%, CD4 median 179 Raby et al, Plos ONE 2008 59 55 75 T cell anergy in 17%, CD4 median 212 Aabye et al, Plos ONE 2009 68 83 T cell anergy in 22%, CD4 median 179 Rangaka et al, CID 2007 31 67 90 T cell anergy no reported, No commercial test, CD4 median 167

Liebeschuetz et al, Lancet 2004 Comparison of TST/IGRAs in patients with active TB microbiologically confirmed Source Patient number TST+ % T-SPOT TB+ QTF-G+ RD1 proteins ELISPOT+ % To note Liebeschuetz et al, Lancet 2004 57 35 81 nd T cell anergy no reported, CD4 nd Vincenti et al, Clin Exp Imm 2007 13 46 85 69 T cell anergy in 20%, CD4 median 179 Raby et al, Plos ONE 2008 59 55 75 63 T cell anergy in 17%, CD4 median 212 Aabye et al, Plos ONE 2009 68 83 65 T cell anergy in 14%, CD4 median 179 Rangaka et al, CID 2007 31 67 90 T cell anergy no reported, No commercial test, CD4 median 167

Proportion of in vitro anergic responses to IGRAs in HIV+ patients Brock, Resp Res 2007 Vincenti, Clin Exp Imm 2007 Luetkemeyer, AJRCCM 2007 Clark, Clin Exp Imm 2007 Karam, Plos ONE 2008 Rabi, Plos ONE 2008 Test QFT ELISPOT home-made N. Paz 590 111 196 201 247 84 CD4 per ml <100 4 (24%) 12 (57%) 5 (16%) 4 (6%) 6 (16%) 6 (46%) 100-200 1 (3%) 4 (19%) 4 (3.6%) 1 (NA) 12 (31%) 3 (15%) 201-300 5 (8%) 3 (14%) 10 (26) 3 (13%) >300 10 (2%) 2 (10%) 6 (3.9%) 8 (21%) 4 (8%)

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

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

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

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

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

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests 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-g Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

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 Et encore: pourquoi est-il important de différencier la phase active de la phase latente de la TB? Premièrement Pour fournir un diagnostic correcte: étant donné que la maladie active conduit à la destruction de l’organe interressé, tandis que l’infection non. deuxièmement Pour pouvoir choisir un traitement correct. En effet dans le cas de la tuberculose active la thérapie doit etre de 6 mois, 2 mois avec 4 medicaments et 4 mois avec 2 medicaments, par contre dans le cas de l’infection latente le traitement et de 6 mois avec 1 seul medicament. Troisièmement. Pour un bon équilibre coût/bénéfice en évitant des procédures complexes( radiologiques, cliniques, et chirurgicales) Ex: TB extra-pulmonaire

DR-serological specificities covered Our approach: use of peptides from ESAT-6 and CFP-10 selected by computational analysis Peptide Position sequence DR-serological specificities covered 1- ESAT-6 6-28 1, 3, 4, 8, 11(5), 13(6), 52, 53 2- ESAT-6 66-78 3, 8, 11(5), 13(6), 15(2), 52 3- CFP-10 18-31 3, 5, 11(5), 52 4- CFP-10 43-70 1, 3, 4, 7, 8, 11(5),13(6), 15(2), 52 5- CFP-10 74-86 3, 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

IFN-g response to RD1 selected peptides is associated to active TB Meeting INMI - Biomerieux • Diagnosis of Active & Latent TB August 25th, 2005 IFN-g response to RD1 selected peptides is associated to active TB Differently using selected peptides we demonstrated that the IFN-gamma response to these antigens evaluated by ELISPOT is specifically associated to active TB. In fact on those with active TB indicated in white a positive response is present to both intact and selected peptides, whereas those with latent TB, TST positive, indicated in red, recognize only intact proteins, no selected peptides. Thus this assay discriminates between latent and active disease. In addition this assay may help to monitor therapy efficacy because the peptides response goes under detection in those responding to anti-TB therapy(iwhite lines), whereas is still detected in those not responding to therapy (red lines). Vincenti et al, Mol Med 2003 INMI, Goletti 51

In patients with active TB the response to RD1 selected peptides decreases after efficacious treatment Differently using selected peptides we demonstrated that the IFN-gamma response to these antigens evaluated by ELISPOT is specifically associated to active TB. In fact on those with active TB indicated in white a positive response is present to both intact and selected peptides, whereas those with latent TB, TST positive, indicated in red, recognize only intact proteins, no selected peptides. Thus this assay discriminates between latent and active disease. In addition this assay may help to monitor therapy efficacy because the peptides response goes under detection in those responding to anti-TB therapy(iwhite lines), whereas is still detected in those not responding to therapy (red lines). Carrara et al, CID 2004

RD1 commercially available assays Sensitivity, specificity and diagnostic odds ratio of the different assays for the immune diagnosis of TB Assay Sensitivity % Specificity Diagnostic odds ratio RD1 ELISPOT assays Selected peptides 70 91 22 Intact proteins 83 56 6 RD1 commercially available assays T-SPOT TB 59 15 QFT Gold 7 Sensitivity of our in house assays based on selected peptides was lower compared to the other assays; however the specificity was much higher and more importantly the diagnostic odds ratio was significantly higher compared to the other tests Goletti et al, CMI 2006

TBNET report Goletti et al, PLoS ONE 2008

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

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

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests 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-g Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

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

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

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests 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-g Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

IP-10 is induced by ESAT-6, CFP10 e TB7 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-g detection of the QFT-Gold Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

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

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

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests 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-g Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests 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-g Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

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

Agenda Problems in the diagnosis of TB TST IGRA New experimental tests 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-g Je vous parle d’un nouveau test pour le diagnostic de la tuberculose. Pourquoi? Qu’est-ce que c’est la TB? Et pourquoi a t-on besoin d’un nouveau test? Pour répondre a ces questions j’ai préparé une liste d’objectifs.

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