Istituto Nazionale per le malattie Infettive L. Spallanzani Roma, Italy Immunodiagnosis of TB Delia...

Istituto Nazionale per le malattie Infettive L. SpallanzaniRoma, Italy

Immunodiagnosis of TB

Delia Goletti

Borstel, May 28th, 2010

Agenda

Problems in the diagnosis of TB

TST

IGRA QuantiFERON-TB RD1-based assays:

T-SPOT TB QuantiFERON-TB Gold

New experimental tests

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


TST




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

Positive TST

M. tuberculosis

Active TB disease

Latent TB infection

NTM Exposure to environmental mycobacteria

BCG-vaccination

BCG-vaccination

Tuberculin skin test (TST)-2

TST does not distinguishamong 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

Agenda


TST




QuantiFERON®-TB

Nil

Tuberculin PPD

M. Avium PPD

Mitogen

QuantiFERON®-TB Test Method (in 2002...)

Heparinised whole blood

AvianPPD

TuberculinPPD

MitogenControl

Transfer undiluted whole bloodinto wells of a culture plate

and add antigens

Culture overnight at 37oC

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/mlO

D 4

50n

m

Standard Curve

Measure OD anddetermine IFN- levels

Stage 1: Whole Blood CultureNil

Control

Stage 2: IFN-ELISA

16

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…




Agenda


TST




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


TST

QuantiFERON-TB

RD1-based assays: T-SPOT TB QuantiFERON-TB Gold


T-SPOT TB

Lalvani et al, JID 2001

ESAT-6: 17 peptides

CFP-10: 18 peptides

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-

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


TST




QuantiFERON®-TB Gold In tube: peptides used

Mori et al, AJRCCM 2004

ESAT-6: 7 peptides

CFP-10: 6 peptides

TB7.7: 1 peptide

QuantiFERON®-TB Gold In tube: methods

Collect peripheral venous blood

(4 or 8 ml)

Harvest plasmaKeep 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

Stage 1:

Culture overnight at 37oC

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

TMBTMB

COLORCOLOR

Measure OD anddetermine IFN- levels

Stage 2:

IFN- ELISA

IFN-IU/mlOD

45

0n

mO

D 4

50

nm

Standard CurveStandard Curve

QuantiFERON®-TB Gold In tube

Comparison TST vs IGRATST 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 CD4 T cells in vitro

Cytokines involved

IFN-, TNF-, TNF- IFN- IFN-

Modified from Mack et al, ERJ 2009

Comparison TST vs IGRA

TST RD1 IGRA



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


Comparison TST vs IGRATST RD1 IGRA



IT)

Internal control no yes yes

Technical expertise required Medium high

Medium high Low medium

Cost of reader machine - Medium high Low medium

Cost of the assay 2-3 euros

30-35 euros? 30-35 euros?


Accuracy

TST

T-SPOT TB

QuantiFERON-TB Gold

TST sensitivity

SENSITIVITY TST 77%Pai et al, Annals 2008

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 T SPOT TB 90%

SENSITIVITY QFT-IT 70%

Specificity T-SPOT TB vs QuantiFERON TB-Gold In tube


SPECIFICITY T SPOT TB 93%

SPECIFICITY QFT-IT 96%

Positive RD1-IGRA

Positive M. tuberculosis infection/diseas

e

NTM

BCG-vaccination

RD1-IGRA

Positive RD1-IGRA do not distinguishactive TB disease and LTBI

Active TB disease

Latent TB infection

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 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

Comparison of TST/IGRAs in patients HIV+ with active TB microbiologically confirmed


TST+

%

T-SPOT TB+

%

QTF-G+

%

RD1 proteins ELISPOT+

%

To note


57 35 81 nd nd T cell anergy no reported, CD4 nd

Vincenti et al, Clin Exp Imm 2007

13 46 85 85 nd T cell anergy in 20%, CD4 median 179

Raby et al, Plos ONE 2008

59 55 nd 75 nd T cell anergy in 17%, CD4 median 212

Aabye et al, Plos ONE 2009

68 nd nd 83 nd T cell anergy in 22%, CD4 median 179

Rangaka et al, CID 2007

31 67 nd nd 90 T cell anergy no reported, No commercial test, CD4 median 167

Comparison of TST/IGRAs in patients with active TB microbiologically confirmed


TST+

%

T-SPOT TB+

%

QTF-G+

%

RD1 proteins ELISPOT+

%

To note


57 35 81 nd nd T cell anergy no reported, CD4 nd

Vincenti et al, Clin Exp Imm 2007

13 46 85

69

85

69

nd T cell anergy in 20%, CD4 median 179

Raby et al, Plos ONE 2008

59 55 nd 75

63


Aabye et al, Plos ONE 2009

68 nd nd 83

65


Rangaka et al, CID 2007

31 67 nd nd 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

QFT QFT ELISPOT home-made

QFT

N. Paz 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%)

>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…




Agenda Problems in the diagnosis of TB

TST



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-

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

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- response to RD1 selected peptides is associated to active TB

Vincenti et al, Mol Med 2003

LTBI - ACTIVE TB0

100

200

300

400CTRPHAPPDRD1 proteinsRD1 peptidesIF

N-

(SF

C p

er m

illi

on

PB

MC

)

In patients with active TB the response to RD1 selected peptides decreases after efficacious treatment

Carrara et al, CID 2004

0 1 2 3 40

100

200No responders toanti-TB therapy

Responders to anti-TBtherapy

0 3Months after anti-TB therapy

IFN

- S

FC

/p

er m

illi

on

PB

MC

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

91 59 15

QFT Gold 83 59 7

Goletti et al, CMI 2006

TBNET report

Goletti et al, PLoS ONE 2008

In vitro IFN- 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


TST



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-

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 2007PLEURAL CELLSPBMC


TST




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

Detection of IP-10 in the plasma harvested from QuantiFERON-TB Gold In-tube

IFN-

Controls TB0

250

500

750

1000

1250p

g/m

l

IP-10

Controls TB0

500

1000

1500

pg

/ml

IP-10 detected in samples from patients with active TB with a negative QFT results

Controls TB0

100

200

300

400

500

600

pg

/ml

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


TST




Dynamic relationship between IFN- and IL-2 profile of M. tuberculosis-specific T cells and antigen load

0

25

50

75

100

IFN-

IL-2

IFN-/IL-2

Active TB diagnosis End of therapy

tota

l cy

toki

ne-

secr

etin

gR

D1-

spec

ific

CD

4+ T

cel

ls (

%)

From Millington, J Immunol 2007, modified


TST




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Istituto Nazionale per le malattie Infettive L. Spallanzani Roma, Italy Immunodiagnosis of TB Delia...

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