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May 23-26, 2012 in Bucharest, Romania “TB and M/XDR-TB: from clinical management to control and elimination” ERS School IGRA testing to diagnose TB disease and infection: What is new in clinical practice and for programmatic management? Delia Goletti Translational Research Unit, INMI, Italy Martina Sester Department of Transplant and Infection Immunology Saarland University; Germany

Agenda LTBI definition TST IGRA New experimental tests

Agenda LTBI definition TST IGRA New experimental tests

Different stages of tuberculosis Infection eliminated with or without T cell priming Infection (latent tuberculosis infection, LTBI) – Recent ( with half of the total risk to progress to active disease within 2 years ) – Latent ( with half of the risk to progress to active disease during the whole life time )  Active disease Bacterial load ? Young et al, Trends in Immunol, 2009 Barry et al, Nature Reviews Microbiol, 2009

Latent infection with M. tuberculosis Direct identification of M. tuberculosis in individuals who are latently infected is not possible. LTBI is a status characterized by the absence of clinical, and radiological evidence of TB disease and the diagnosis is assessed by the presence of an M. tuberculosis-specific immune responses due to: – a presumptive infection with M. tuberculosis without the presence of living bacteria – a presumptive persistence of living M. tuberculosis in a state of altered metabolism that potentially may reactivate later Mack et al, ERJ 2009

Evidence for the existence of LTBI? TST + contacts have a higher risk for developing TB that is reduced by INH treatment Erkens et al. Eur Respir J 2010 Treatment regimenEfficacy/effectivenessEvidence 12 mo INH93-75%A 9 mo INH90%C 6 mo INH69-65%A 4 mo RIFunknown (>3 mo INH/RIF)C 3 mo INH/RIFequivalent to 6 mo INHA

Latent infection with M. tuberculosis: size of the problem It is estimated (by TST) that 2 billion people globally are latently infected with M. tuberculosis LTBI subjects may develop active TB because of the waning of effective host immune responses due to: – chronic diseases such as diabetes, alcoholic liver disease, renal failure – malnutrition – immunosuppression HIV co-infection immunosuppressive drugs

Agenda LTBI definition TST IGRA New experimental tests

in vivo PPD IFN  TNF  Chemokine Tuberculin (PPD) Tuberculin Skin Test (TST)

in vivo PPD IFN  TNF  Chemokine Tuberculin (PPD) Tuberculin Skin Test (TST) Drawbacks 48-72h duration Falsly positive after BCG vaccination Low sensitivity in immunocompromised patients Variablitity in reading of test result

Positive TST M. tuberculosis Active TB disease Latent TB infection Non Tuberculous Mycobacteria (NTM) Exposure to environmental mycobacteria or disease BCGBCG-vaccination TST TST does not distinguish among all these different clinical situations

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

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

Species specificities of mycobacterial antigens RD-1 encodes ESAT-6 und CFP-10 RD-1 present in M. tuberculosis M. kansasii M. marinum M. szulgai M. flavescens M. leprae (?) RD-1 deletion in M. bovis BCG atypical mycobacteria (e.g. M. avium) Andersen et al Lancet (2000) 356: 1099 RD-1 RD-1 deletion

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--

Agenda LTBI definition TST IGRA New experimental tests

APC T cell antigens/ peptides cytokine induction cytokine induction activation/ cytokine induction cytokine induction ELISAELISPOT assayFlow-cytometry cytokine activation marker Skin test Immunodiagnosis of latent M. tuberculosis infection T.SPOT.TBQuantiFERON TB gold IGRA IFN-  release assay PPD ESAT-6/CFP-10/TB7.7 Negative controls Positive controls, i.e. mitogens PHA/SEB

ELISAELISPOT cut-off: 0,35 IU IFN-  /ml cut-off: >5 SFC/ PBMC - PHA ESAT-6 CFP-10 person A person B ESAT/CFP PHA - ESAT-6 CFP-10PHA-- Incubate 16-24h Incubate 16-24h

Examples of test results Nil MitogenESAT-6CFP-10 positive negative indeterminate negative

Positive IGRA BCG-vaccination NTM Positive M. tuberculosis infection/disease IGRA results Nil MitogenESAT-6CFP-10

Comparison TST vs IGRA TST ELISPOT (T-SPOT TB) ELISA (QuantiFERON-TB Gold IT) Internal controlnoyes AntigensPPD Peptides from CFP-10, ESAT-6 Peptides from CFP-10, ESAT-6 and TB7.7 Tests’ substrateSkinPBMCWhole 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- 

Comparison TST vs IGRA TST ELISPOT (T-SPOT TB) ELISA (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 Mack et al, ERJ 2009

Comparison TST vs IGRA TST ELISPOT (T-SPOT TB) ELISA (QuantiFERON-TB Gold IT) Technical expertise required Medium high Low medium Cost of reader machine -Medium highLow medium Cost of the assay 2-3 euros30-35 euros? Mack et al, ERJ 2009

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

Metaanalysis of IGRA to diagnose active TB 817 excluded in total for the following reasons:  lab studies233  other than QFT-G-IT or T-SPOT.TB169  no original article152  animal studies123  TB not confirmed109  under treatment12  not according to manufacturer instructions11  cutoffs not used in Europe1  manuscript not available1  non-tuberculosis patients1  duplicate studies4  same patients as in other study1 Distributed among 5 pairs of 2 experts 791 excluded 26 excluded 53 studies analyzed in detail 844 potentially relevant citations identified by electronic databases (825) and supplementary sources (19) Distributed among 5 pairs of 2 experts Diagnostic assays:  T-SPOT.TB and TST 11  QFT-G-IT and TST4  T-SPOT.TB, QFT-G-IT and TST 4  Other IGRA combinations8 Origin of study:  Low prevalence country15  High prevalence country12 27 studies finally included  blood 18  extrasanguinous9 Sester, Sotgiu et al. Eur Respir J (2011), 37:

Test SensitivitySpecificity Diagnostic Odds Ratio TST QFT-G-IT blood extrasang T-SPOT.TB blood extrasang Sester, Sotgiu et al. Eur Respir J (2011), 37: Metaanalysis of IGRA to diagnose active TB -summary of pooled values-

Conclusions of metaanalysis Sensitivities of both IGRAs in detecting active TB were higher than that of TST – Sensitivities of IGRAs are not high enough to be used as rule out tests for tuberculosis Specificity of IGRAs is insufficient when assessed among controls including TB suspects – No distinction between active TB and latent M. tuberculosis infection Highest sensitivity and diagnostic OR when using T-SPOT.TB from extrasanguinous fluids (e.g. BAL) Sester, Sotgiu et al. Eur Respir J (2011), 37:

Positive IGRA BCG-vaccination NTM Positive M. tuberculosis infection/disease IGRA results Nil MitogenESAT-6CFP-10 Active TB disease Latent TB infection: Recently or remotely acquired Positive RD1-IGRA do not distinguish active TB disease and LTBI

Importance to distinguish between latent infection and active TB To provide a correct diagnosis – Active TB 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 then 4 months therapy with 2 drugs – Latent infection 6 months therapy with one drug To save human and economic costs avoiding complex evaluations

Agenda LTBI definition TST IGRA New experimental tests

Antigen different from the commercial RD1 peptides -Rv3615, RD1 selected peptides, antigens of latency, Rv2628, HBHA Marker different from IFN-  -IP-10, MCP-2, IL-2 Readout different from ELISA or ELISPOT Biological sample different from blood -BAL, pleural fluid, urine, CNS

Rv3615c as a RD1-secreted antigen specific for M. tuberculosis infection Millington et al, PNAS 2011 Active TBLTBI

Use of ESAT-6/CFP-10 peptides selected by computational analysis Peptides selected by computational analysis that cover more than 90% of the HLA class II specificities 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) Goletti et al, CDLI 2005

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

Response to RD1 selected peptides decreases after efficacious treatment Carrara et al, CID 2004

IFN-  response to latency antigen Rv2628 is associated to remote LTBI Goletti et al, ERJ 2010

Response to Rv2628 is increased at the site of TB disease in active TB Chiacchio et al, Plos One 2011

The frequency of the RD1 response is higher compared to that to Rv2628 Chiacchio et al, Plos One 2011

Specific T-cells are predominantly monofunctional in BAL and peripheral blood Chiacchio et al, Plos One 2011

Proportions of EM cells Increased proportion of EM in BAL compared to peripheral blood in response to RD1 Increased proportion of EM in response to RD1 compared o Rv2628 in BAL Chiacchio et al, Plos One 2011

Rv2628-response in peripheral blood is associated to remote LTBI Screening of contacts of patients with active TB, after exclusion of active TB, among those positive to IGRA IGRA-positive Rv2628+Rv2628- Likely Remote LTBI Likely Recent Infection Higher need of chemoprophylaxis

IFN-  response to the methylated HBHA of M. tuberculosis produced in M. smegmatis is reduced in patients with active TB Delogu, et al and Goletti, PloS One 2011

Response to HBHA of M. tuberculosis produced from M. smegmatis is mediated by effector memory CD4+ T cells 7% 84% 4% 5% CD62L APC-A CD45RO PE-Cy7-A 0% 45% 55% IFN-γ FITC-A CD4 APC H7-A 0% 83% 17% CD8 PerCP Cy5.5-A IFN-γ FITC-A 0% 0.07% 46% 53% IFN-γ FITC-A CD4 APC H7-A 0.07% 0% 83% 17% CD8 PerCP Cy5.5-A IFN-γ FITC-A A B C DE control HBHA Delogu, et al and Goletti, PloS One 2011

Response to rHBHAms is significantly impaired in patients with active TB Delogu, et al and Goletti, PloS One 2011

IFN-  response to rHBHAms ROC analyses AUC 0.72 (CI ) Sensitivity 50% Specificity 80% Cut-off: 0.25U/ml AUC 0.78 (CI ) Sensitivity 75% Specificity 75% Cut-off: 0.75U/ml AUC 0.62 (CI ) Delogu, et al and Goletti, PloS One 2011

Lack of recovery of response to HBHA of M. tuberculosis produced from M. smegmatis in active TB Delogu, et al and Goletti, PloS One 2011

Response to methylated HBHA of M. tuberculosis is associated with TB control Screening of subjects suspected of active TB, among those positive to IGRA IGRA-positive mHBHA-mHBHA+ Likely active TB Likely no active TB Recent Infection, remote Infection, past cured TB

New experimental tests Antigen different from the commercial RD1 peptides -Rv3615, RD1 selected peptides, antigens of latency, Rv2628, HBHA Marker different from IFN-  -IP-10, MCP-2, IL-2 Readout different from ELISA or ELISPOT Biological sample different from blood -BAL, pleural fluid, urine, CNS

IP-10 induced by ESAT-6, CFP10, and TB7.7 in patients with TB disease QFT-Gold, detection of: IP-10 (Ruhwald, 2007): – Significantly higher in patients with active disease – IP-10 detectable in patients with active TB scored negative by IFN-  detection of the QFT-Gold

IP-10 and MCP-2 are associated with active TB Ruhwald et al, ERJ 2008

IP-10 response in HIV-infected subjects in India Goletti et al, PLoS One 2010

IFN-  induction is impaired in HIV + patients defined as “mitogen- unresponsive” Goletti et al, PLoS One 2010

IP-10 vs IFN-  response overtime: QFT-IT Kabeer et al, BMC ID 2011 IP-10 vs IFN-γ

IP-10 vs IFN-  response overtime: RD1 selected peptides-based assay Kabeer et al, BMC ID 2011 IP-10 vs IFN-γ

New experimental tests Antigen different from the commercial RD1 peptides -Rv3615, RD1 selected peptides, antigens of latency, Rv2628, HBHA Marker different from IFN-  -IP-10, MCP-2, IL-2 Readout different from ELISA or ELISPOT Biological sample different from blood -BAL, pleural fluid, urine, CNS

Loss of PPD-specific IFN-γ/IL-2 dual-positive T cells in active TB Sester et al, Plos One 2011 A-TB, n=25 T-TB, n=28 IFN  /IL-2 pos. <56% Specificity: 100% Sensitivity: 70%

Dominance of dual-positive CD4 T cells in other non-active states BCG-vaccinated, n=25 IFN  /IL-2 pos. <56% Specificity: 100% Sensitivity: 70% Latent infection, n=25 Sester et al, Plos One 2011

Dominant TNF-  CD4 T cell responses discriminate between LTBI and active TB Harari et al, Nature medicine 2011 Decrease in multifunctionality Increase in single functionality

Dominant TNF-  response in active TB n=76 latent, n=18 active TNF-  single pos. >37.4% Specificity: 96.1% Sensitivity: 66.7% Harari et al, Nature medicine 2011

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

New experimental tests Antigen different from the commercial RD1 peptides -Rv3615, RD1 selected peptides, antigens of latency, Rv2628, HBHA Marker different from IFN-  -IP-10, MCP-2, IL-2 Readout different from ELISA or ELISPOT Biological sample different from blood -BAL, pleural fluid, urine, CNS

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

IGRA at the site of TB disease: pleural fluid vs blood Losi et al, ERJ 2007 PLEURAL CELLSPBMC

Chemokines in urine: IP-10 is increased in patients with active TB Cannas et al, BMC ID 2010

IP-10 is increased in the urine of patients with lung disease Cannas et al, BMC ID 2010

IP-10 decreases in the urine of TB patients after successful therapy Cannas et al, BMC ID 2010

Skin test based on rdESAT-6 in humans infected with M. tuberculosis Arend et al, Tuberculosis 2007

Acknowledgements…