Istituto Nazionale per le malattie Infettive L. Spallanzani Roma, Italy Diagnosis of latent...

Istituto Nazionale per le malattie Infettive L. SpallanzaniRoma, Italy

Diagnosis of latent tuberculosis infection: the role of IGRAs

Delia Goletti, MD, PhD

Translational Research Unit, INMI

ERS online course on tuberculosis, March 8th, 2011

March 8th , 2011 Best wishes to all women in the “woman

day”!

Agenda

LTBI definition

TST

IGRA

New experimental tests

Global burden of TB in 2009

Estimated number of

CasesN. (%)

DeathsN. (%)

All forms of TBWomen

9.4 million3.3 million (35%)

1.3 million380,000 (29%)

HIV-associated TB

1.1 million (12%) 400,000 (36%)

MDR-TB 440,000 (4.6%) 150,000 (34%)

WHO report, 2010

Immunity against M. tuberculosis

Adapted by Schwander and Dheda, 2011

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 ?

Adapted from: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 performed by an immune test that ascertains M. tuberculosis-specific immune responses (positive TST or an IGRA) due to:

a presumptive infection with M. tuberculosis (Mack et al, ERJ 2009)

a condition where human tissues contain living M. tuberculosis that persists in a state of altered metabolism that potentially may later reactivate (Opie and Aronson, 1927; Ulrichs et al, JID 2005)

Evidence for the existence of LTBI?

TST+ contacts have a higher risk for developing TB that is reduced by INH treatment (Ferebee et al, 1962; Veening et al, 1968; Egsmose et al, 1965)

Erkens C et al. Eur Respir J 2010

Treatment regimen Efficacy/effectiveness Evidence

12 mo INH 93%/75% A

9 mo INH 90% C

6 mo INH 69%65% A

4 mo RIFunknown (>3 mo

INH/RIF)C

3 mo INH/RIF equivalent to 6 mo INH A

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 (Sudre et al, 1992)

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, malnutrition, immunosuppression due to :

HIV co-infection steroids or other immunosuppressive drugs

Agenda

LTBI definition

TST

IGRA


Principle of the Tuberculin skin test (TST)

intradermal antigen-inoculation

uptake by antigen-presenting cells

antigen-presentation in lymph nodes interaction with T-cells

cytokine-release clonal T-cell expansion

increase in capillary permeability

influx of memory T cells into the test area

palpable induration (max. at 48-72 h)

Limitations of the TST 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

Positive TST

M. tuberculosis

Active TB disease

Latent TB infection

Non Tuberculosis Micobacteria

(NTM)

Exposure to environmental mycobacteria or disease

BCG-vaccination

BCG-vaccination

TST

TST does not distinguishamong all these different clinical situations

Need of…

Standardized test (laboratory test)

M. tuberculosis-specific reagents

Possibility to discriminate between the different stages of tuberculosis

Species specificities of ESAT-6 and CFP-10

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 - -

Tuberculosis complex

Antigens

ESAT CFP

M tuberculosis + +M africanum + +M bovis + +BCG substrain

gothenburg - - moreau - - tice - - tokyo - - danish - - glaxo - - montreal - - pasteur - -

Agenda

LTBI definition

TST

IGRA


IGRA

RD1

IFN-γ

PBMC Whole Blood

T SPOT.TB QuantiFERON TB Gold In tube

IGRA

Nil (negative control)

RD1 peptides (M. tuberculosis-specific antigens)

Mitogen (positive control)Test Result Nil RD1 peptides

(M. tuberculosis-specific antigen)

Mitogen

Indeterminate

– – –

Negative – – +

Positive – + +

Positive RD1-IGRA

BCG-vaccination

NTM

Positive M. tuberculosis infection/disease

RD1-IGRA

Comparison TST vs IGRATST RD1 IGRA

ELISPOT

(e.g. T-SPOT TB)

ELISA

(e.g. QuantiFERON-TB Gold IT)

Internal control no yes yes

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 CD4 T cells in vitro

Cytokines involved

IFN-γ, TNF-α, TNF-β

IFN-γ IFN-γ

Modified from Mack et al, ERJ 2009


ELISPOT

(e.g. T-SPOT TB)

ELISA


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



ELISPOT

(e.g. T-SPOT TB)

ELISA


Technical expertise required

Medium high Medium high Low medium

Cost of reader machine

- Medium high Low medium

Cost of the assay

low high high



ELISPOT

(e.g. T-SPOT TB)

ELISA


Conversion Criteria established for recent infection

Not established yet Not established yet

Recent vs remote infection

Does not differentiate

Does not differentiate Does not differentiate

Exposure correlation

Some degree, especially if not BCG-vaccinated

high high

Modified from Leung et al, ERJ 2011

Need of…


M. tuberculosis-specific reagents: accuracy


Accuracy

Sensitivity and specificity

Predictive value of IGRA for active TB development

Efficacy of preventive therapy based on IGRA results

Accuracy of IGRA: sensitivity and specificity

Summary of pooled values from the metanalysis performed by Pai et al, and by Sester and Sotgiu et al

TestSensitivity for active

TB

Specificity for TB infection

Specificity for active TB

Percentage

TST Pai et al, 2008 77 59/97

Sester et Sotgiu et al,

201065 75

QFT-IT

Pai et al, 2008 70 96 -


201080 - 79

T-SPOT.TB

Pai et al, 2008 90 93 -


201081 -

59

Conclusions 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

Accuracy



Efficacy of preventive therapy based on IGRA results

Predictive value of IGRA: HIV-negative subjects

Diel et al, AJRCCM 2010

1414 contacts followed in Hamburg, Germany

Negative predictive value of T-SPOT. TB assay in tuberculosis suspects

Diel , Goletti et al, ERJ 2010

Negative predictive value for progression in QuantiFERON-TB Gold In-Tube or T-SPOT.TB assay negative subjects

Diel , Goletti et al, ERJ 2010

Rates of disease progression in IGRA+ vs TST +

Country Test Incidence of active TB in IGRA+ groups

Comment

Gambia [Hill et al. 2008]

ELISPOT (in-house)

9/1000 person-yr High burden

Colombia [del Corral et al. 2009]

In-house CFP-10 assay

7/1000 person-yr High Burden

Senegal [Lienhardt et al. 2010]

ELISPOT (in-house- 32 SFC cut-point)

9/1000 person-yr High burden

Turkey [Bakir et al. 2008]

ELISPOT (in house similar to T-SPOT TB)

21/1000 person-yr Intermediate

Germany [Diel et al. 2010]

QFT-IT 73/ 1000 person-yr Low burden

From Pai and Dheda, personal data 2011

Accuracy



Efficacy of preventive therapy based on IGRA results: .......................NO DATA AVAILABLE....................

Vulnerable populations

Children

Immuno-suppressed for: HIV Autoimmune disease

Sensitivity and specificity of IGRAs in children with active TB

Ling at al, Paediatric Respiratory Reviews, 2011

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 57 81 NA TB microbiologically diagnosed

Kampmann et al, ERJ 2009

25 83 58 80 TB microbiologically diagnosed

Hermann JL et al, Plos 2008

32 87 NA 78 TB microbiologically diagnosed in 48%

Nicol et al, Pediatrics 2009

10 80 50 NA TB microbiologically diagnosed

Connell et al, Plos 2008

9 89 100 89 TB clinically diagnosed

IGRA in HIV+ with active TB, as a surrogate marker for the accuracy in LTBI

Hoffmann and Ravn, European Infectious Diseases, 2010

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

QFT QFT ELISPOT home-made

QFT

N. Patients

590 111 196 201 247 84

CD4 per l

<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%)

IGRAs in subjects with for autoimmune diseases under immune suppressive therapy

Solovic et al, ERJ 2010

Positive RD1-IGRA

BCG-vaccination

NTM

Positive M. tuberculosis infection/diseas

e

RD1-IGRA

Positive RD1-IGRA do not distinguishactive TB disease and LTBI

Active TB disease

Latent TB infection:

• Recently acquired or

• Remotely acquired

Need of…


M. tuberculosis-specific reagents


Agenda

LTBI definition

TST

IGRA


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


Antigen different from the commercial RD1 peptides (RD1 selected peptides, antigens of latency, Rv2628, HBHA)

Marker different from IFN-γ (IP-10, MCP-2, IL-2)

Readout different from ELISA or ELISPOT Biological sample different from blood (BAL,

pleural fluid, CNS)

Our approach: use of peptides from ESAT-6 and CFP-10 selected by computational analysis

Peptides selected by computational analysis that cover more than 90% of the HLA class II specificities

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)

IFN-γ response to RD1 selected peptides is associated to active TB

Modified Vincenti

et al, Mol Med 2003

IFN-γ

responseto the

antigen of latency

Rv2628 is associated to remote LTBI

Goletti et al, ERJ 2010

IFN-γ response to the antigen of latency Rv2628 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 chemoprophilaxis

IFN-γ

responseto the

methilated HBHA of M. tuberculosis

produced in M. smegmatisis

significantly

reduced in patientswithactive

tuberculosis

Delogu, et al and Goletti, in press PloS One

IFN-γ response to the methilated HBHA of M. tuberculosis produced in M. smegmatis is associated with a status of TB control

Screening of subjects suspected of active TB, among those positive to IGRA

IGRA-positive

mHBHA- mHBHA+

LikelyActive TB

LikelyNo active TB:

Recent Infection, Remote Infection,

past cured TB





pleural fluid, CNS)

Detection of IP-10 in the plasma from

QuantiFERON-TB Gold In-tube

From Ruwald et al, modified Microbes Infection 2007

INCREASE OF SENSITIVITY !

IP-10 response in HIV-infected subjects in India

Goletti et al, PLoS One 2010

IFN-γ response to RD1 selected peptides and QFT-IT is impaired in the HIV+ patients defined as “mitogen-unresponsive”

Goletti et al, PLoS One 2010





pleural fluid, CSF)

Dominant TNF-α M. tuberculosis-specific CD4 T cell responses discriminate between LTBI and active TB disease

From Harari et al, Nature medicine 2011





pleural fluid, CSF, skin)

IGRA at the site of TB disease: BAL vs blood

From Jafari, AJRCCM 2009

Skin test based on rdESAT-6 in humans infected with M. tuberculosis

From Arend et al, Tuberculosis 2007

Agenda

LTBI definition

TST

IGRA


And thank you to:

Translational Research Unit, INMI

Outpatient Clinic of Pneumology, INMI

Acknowledgments Epidemiology Department, INMI, Rome, Italy

E. Girardi, G. Ippolito

Translational Research Unit, INMI, Rome, Italy V. Vanini, T. Chiacchio, G. Cuzzi, E. Petruccioli, L. Petrone, D. Goletti

Clinical Department, INMI, Rome, Italy M. Vecchi, C. Copertino, F. Lauria

International collaborations Dept. of Infectious Diseases and Dept. of Immunohematology & Blood Transfusion,

Leiden, The Netherlands K.L.M.C. Franken, T.H.M. Ottenhoff

Case Western Reserve UniversityCleveland, Ohio Zahra Toossi, MD

Hopital Saint Louis de Paris Paris, France Philippe Lagrange, MD

Universidad Autonoma de Barcelona, Barcelona, Spain Josè Dominguez, PhD

Medical Clinical Infectious Diseases Research Center, Borstel, Germany Christoph Lange, MD, PhD

Tuberculosis Research Center (TBRC) Chennai, India Raja Alamelu, PhD

Hinduja Hospital University Medical Center Mumbai, India Camilla Rodriguez, MD

Istituto Nazionale per le malattie Infettive L. Spallanzani Roma, Italy Diagnosis of latent...

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