Post on 31-Mar-2015
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
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
Problems in the diagnosis of TB
TST
IGRA QuantiFERON-TB RD1-based assays:
T-SPOT TB QuantiFERON-TB Gold
New experimental tests
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
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 QuantiFERON-TB RD1-based assays:
T-SPOT TB QuantiFERON-TB Gold
New experimental tests
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…
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 QuantiFERON-TB RD1-based assays:
T-SPOT TB QuantiFERON-TB Gold
New experimental tests
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
Problems in the diagnosis of TB
TST
QuantiFERON-TB
RD1-based assays: T-SPOT TB QuantiFERON-TB Gold
New experimental tests
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
Problems in the diagnosis of TB
TST
IGRA QuantiFERON-TB RD1-based assays:
T-SPOT TB QuantiFERON-TB Gold
New experimental tests
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
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
Comparison TST vs IGRATST RD1 IGRA
ELISPOT (e.g. T-SPOT TB)
ELISA (e.g. QuantiFERON-TB Gold
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?
Modified from Mack et al, ERJ 2009
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
Pai et al, Annals 2008
SENSITIVITY T SPOT TB 90%
SENSITIVITY QFT-IT 70%
Specificity T-SPOT TB vs QuantiFERON TB-Gold In tube
Pai et al, Annals 2008
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
Source Patient number
TST+
%
T-SPOT TB+
%
QTF-G+
%
RD1 proteins ELISPOT+
%
To note
Liebeschuetz et al, Lancet 2004
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
Source Patient number
TST+
%
T-SPOT TB+
%
QTF-G+
%
RD1 proteins ELISPOT+
%
To note
Liebeschuetz et al, Lancet 2004
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
nd T cell anergy in 17%, CD4 median 212
Aabye et al, Plos ONE 2009
68 nd nd 83
65
nd T cell anergy in 14%, CD4 median 179
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…
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 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-
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
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-
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
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-
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
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-
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-
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
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-
And thank you to: