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 2012 by the author
“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
May 23-26, 2012 in Bucharest, Romania
Agenda
•
•
•
•
LTBI definition
TST
IGRA
New experimental tests
Agenda
•
•
•
•
LTBI definition
TST
IGRA
New experimental tests
Different stages of tuberculosis
Bacterial load ?
• 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
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
Treatment regimen
Efficacy/effectiveness
Evidence
12 mo INH
93-75%
A
9 mo INH
90%
C
6 mo INH
69-65%
A
4 mo RIF
unknown (>3 mo INH/RIF)
C
3 mo INH/RIF
equivalent to 6 mo INH
A
Erkens et al. Eur Respir J 2010
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
Tuberculin Skin Test (TST)
IFNg
PPD
in vivo
Tuberculin (PPD)
TNFa
Chemokine
Tuberculin Skin Test (TST)
IFNg
PPD
in vivo
TNFa
Chemokine
Drawbacks
• 48-72h duration
• Falsly positive after BCG vaccination
• Low sensitivity in immunocompromised
patients
• Variablitity in reading of test result
Tuberculin (PPD)
TST
Active TB disease
M. tuberculosis
Latent TB infection
Positive TST
Non Tuberculous
Mycobacteria
(NTM)
Exposure to environmental
mycobacteria or disease
BCG
BCG-vaccination
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
RD-1 deletion
RD-1 deletion in
M. bovis BCG
atypical mycobacteria
(e.g. M. avium)
Andersen et al Lancet (2000) 356: 1099
Agenda
•
•
•
•
LTBI definition
TST
IGRA
New experimental tests
Immunodiagnosis of
latent M. tuberculosis infection
antigens/
peptides
APC
PPD
ESAT-6/CFP-10/TB7.7
Negative controls
Positive controls, i.e. mitogens
PHA/SEB
Skin test
IFN-g release assay
T cell
cytokine
induction
activation/
cytokine
induction
cytokine
induction
ELISA
ELISPOT assay
QuantiFERON TB gold
T.SPOT.TB
Flow-cytometry
activation marker
cytokine
induction
IGRA
cytokine
ELISA
ELISPOT
Incubate 16-24h
Incubate
16-24h
-
PHA
ESAT-6
CFP-10
person A
person B
cut-off:
0,35 IU IFN-g/ml
cut-off:
>5 SFC/250.000 PBMC
Examples of test results
Nil
Mitogen
ESAT-6
CFP-10
positive
negative
negative
indeterminate
indeterminate
negative
IGRA results
Nil
Mitogen
ESAT-6
CFP-10
BCG-vaccination
NTM
Positive IGRA
Positive M. tuberculosis
infection/disease
Comparison TST vs IGRA
TST
ELISPOT
(T-SPOT TB)
ELISA
(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
Neutrophils, CD4, CD8
that transmigrate out of
capillaries into the skin.
Treg
(CD4+CD25highFoxP3+).
CD4 T cells in vitro
CD4 T cells in vitro
IFN-g, TNF-a, TNF-b
IFN-g
IFN-g
Cells involved
Cytokines involved
Comparison TST vs IGRA
TST
Read-out
Outcomes
measure
Read-out units
ELISPOT
(T-SPOT TB)
ELISA
(QuantiFERON-TB
Gold IT)
Measure of
Measure of optical
Enumeration of IFN-g
diameter of dermal
density values of IFNspots
induration
g production
Level of induration
Number of IFN-g
producing T cells
Plasma concentration
of IFN-g produced by
T cells
mm
IFN-g spot forming
cells
IU/ml
Mack et al, ERJ 2009
Comparison TST vs IGRA
Technical
expertise
required
Cost of reader
machine
Cost of the assay
TST
ELISPOT
(T-SPOT TB)
ELISA
(QuantiFERON-TB
Gold IT)
Medium high
Medium high
Low medium
-
Medium high
Low medium
2-3 euros
30-35 euros?
30-35 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
-summary of pooled valuesTest
Sensitivity
Specificity
Diagnostic
Odds Ratio
TST
0.65
0.75
5.72
0.80
0.79
11.47
extrasang. 0.48
0.82
0.59
0.82
3.84
18.86
35.83
QFT-G-IT
blood
0.81
T-SPOT.TB
extrasang. 0.88
blood
Sester, Sotgiu et al. Eur Respir J (2011), 37: 100-111
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: 100-111
IGRA results
Nil
Mitogen
ESAT-6
CFP-10
BCG-vaccination
NTM
Positive
IGRA
Positive M.
tuberculosis
infection/disease
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
New experimental tests
• Antigen different from the commercial RD1
peptides
-
Rv3615, RD1 selected peptides, antigens of
latency, Rv2628, HBHA
• Marker different from IFN-g
-
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
Active TB
LTBI
Millington et al, PNAS 2011
Use of ESAT-6/CFP-10 peptides
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
Goletti et al, CDLI 2005
IFN-g response to RD1 selected
peptides is associated to active TB
LTBI
Active TB
Modified Vincenti et al, Mol Med 2003
Response to RD1 selected peptides
decreases after efficacious treatment
Carrara et al, CID 2004
IFN-g 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
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-g 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
0%
0%
83%
17%
0%
IFN-γ FITC-A
IFN-γ FITC-A
control
0%
45%
CD8 PerCP Cy5.5-A
55%
CD4 APC H7-A
B
A
83%
0%
0%
17%
46%
CD8 PerCP Cy5.5-A
C
0.07%
IFN-γ FITC-A
IFN-γ FITC-A
0.07%
CD45RO PE-Cy7-A
HBHA
53%
4%
5%
7%
CD62L APC-A
CD4 APC H7-A
D
84%
E
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-g response to rHBHAms
ROC analyses
AUC 0.72 (CI 0.6-0.83)
•Sensitivity 50%
•Specificity 80%
•Cut-off: 0.25U/ml
AUC 0.78 (CI 0.64-0.91)
•Sensitivity 75%
•Specificity 75%
•Cut-off: 0.75U/ml
AUC 0.62
(CI 0.49-0.74)
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-g
-
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-g 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-g induction is impaired in HIV+
patients defined as “mitogenunresponsive”
Goletti et al, PLoS One 2010
IP-10 vs IFN-g response overtime:
QFT-IT
IP-10 vs IFN-γ
Kabeer et al, BMC ID 2011
IP-10 vs IFN-g response overtime:
RD1 selected peptides-based assay
IP-10 vs IFN-γ
Kabeer et al, BMC ID 2011
New experimental tests
• Antigen different from the commercial RD1
peptides
-
Rv3615, RD1 selected peptides, antigens of
latency, Rv2628, HBHA
• Marker different from IFN-g
-
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
T-TB, n=28
A-TB, n=25
IFNg/IL-2 pos. <56%
Specificity: 100%
Sensitivity: 70%
Sester et al, Plos One 2011
Dominance of dual-positive CD4 T cells
in other non-active states
Latent infection, n=25
BCG-vaccinated, n=25
IFNg/IL-2 pos. <56%
Specificity: 100%
Sensitivity: 70%
Sester et al, Plos One 2011
Dominant TNF-a CD4 T cell responses
discriminate between LTBI and active TB
Decrease in multifunctionality
Increase in single functionality
Harari et al, Nature medicine 2011
Dominant TNF-a response in active TB
TNF-a single pos. >37.4%
Specificity: 96.1%
Sensitivity: 66.7%
n=76 latent, n=18 active
Harari et al, Nature medicine 2011
Dynamic relationship between IFN-g 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-g
-
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
PBMC
PLEURAL CELLS
Losi et al, ERJ 2007
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…