Istituto Nazionale per le malattie Infettive L. Spallanzani
Roma, 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
Cases
N. (%)
Deaths
N. (%)
All forms of TB
Women
9.4 million
3.3 million (35%)
1.3 million
380,000 (29%)
HIV-associated TB
1.1 million (12%)
400,000 (36%)
440,000 (4.6%)
150,000 (34%)
MDR-TB
WHO report, 2010
Immunity against M. tuberculosis
Adapted by Schwander and Dheda, 2011
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
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)
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 C 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 (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

New experimental tests
Principle of the Tuberculin skin test (TST)
intradermal antigen-inoculation
uptake by antigen-presenting cells
antigen-presentation in lymph nodes
cytokine-release
interaction with T-cells
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:


Variability:



Purified protein derivative (PPD)
commonly shared among
different Mycobacteria
(M.tuberculosis, BCG and atypical
mycobacteria)
Reproducibility in giving the test
Subjectivity in reading the test
Logistics


Repeat visit needed
3 days before result
TST
Active TB disease
M. tuberculosis
Latent TB infection
Non Tuberculosis
Micobacteria
(NTM)
Exposure to environmental
mycobacteria or disease
BCG-vaccination
BCG-vaccination
Positive TST
TST does not distinguish
among all these different clinical situations
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
Species specificities of ESAT-6 and CFP-10
Tuberculosis
complex
M tuberculosis
M africanum
M bovis
BCG substrain
gothenburg
moreau
tice
tokyo
danish
glaxo
montreal
pasteur
Antigens
ESAT
CFP
+
+
+
+
+
+
-
-
Environmental
strains
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
abcessus
avium
branderi
celatum
chelonae
fortuitum
gordonii
intracellulare
kansasii
malmoense
marinum
oenavense
scrofulaceum
smegmatis
szulgai
terrae
vaccae
xenopi
Antigens
ESAT
CFP
+
+
+
-
+
+
+
-
Agenda

LTBI definition

TST

IGRA

New experimental tests
IGRA
IFN-γ
RD1
PBMC
T SPOT.TB
Whole Blood
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
–
+
+
RD1-IGRA
BCG-vaccination
NTM
Positive RD1-IGRA
Positive M. tuberculosis
infection/disease
Comparison TST vs IGRA
TST
RD1 IGRA
ELISPOT
ELISA
(e.g. T-SPOT TB)
(e.g. QuantiFERON-TB Gold IT)
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
Internal control
Cells involved
Neutrophils, CD4,
CD4 T cells in vitro
CD8 that
transmigrate out of
capillaries into the
skin. Treg
CD4 T cells in vitro
(CD4+CD25highFoxP3+).
Cytokines involved
IFN-γ, TNF-α,
TNF-β
IFN-γ
IFN-γ
Modified from Mack et al, ERJ 2009
Comparison TST vs IGRA
TST
Read-out
Measure of
diameter of dermal
induration
Outcomes measure Level of induration
Read-out units
mm
RD1 IGRA
ELISPOT
ELISA
(e.g. T-SPOT TB)
(e.g. QuantiFERON-TB Gold IT)
Enumeration of IFN-g
spots
Measure of optical
density values of IFN-g
production
Number of IFN-g
producing T cells
Plasma concentration of
IFN-g produced by T
cells
IFN-g spot forming cells
IU/ml
Modified from Mack et al, ERJ 2009
Comparison TST vs IGRA
TST
RD1 IGRA
ELISPOT
ELISA
(e.g. T-SPOT TB)
(e.g. QuantiFERON-TB Gold IT)
Technical
expertise required
Medium high
Medium high
Low medium
Cost of reader
machine
-
Medium high
Low medium
Cost of the assay
low
high
high
Modified from Mack et al, ERJ 2009
Comparison TST vs IGRA
TST
RD1 IGRA
ELISPOT
ELISA
(e.g. T-SPOT TB)
(e.g. QuantiFERON-TB Gold IT)
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…

Standardized test (laboratory test)

M. tuberculosis-specific reagents: accuracy

Possibility to discriminate between the different
stages of tuberculosis
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
Test
Sensitivity for
active TB
Specificity for
TB infection
Specificity for
active TB
Percentage
TST
QFT-IT
T-SPOT.TB
Pai et al, 2008
77
59/97
Sester et Sotgiu
et al, 2010
65
Pai et al, 2008
70
96
-
Sester et Sotgiu
et al, 2010
80
-
79
Pai et al, 2008
90
93
-
Sester et Sotgiu
et al, 2010
81
-
75
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

Sensitivity and specificity

Predictive value of IGRA for active TB development

Efficacy of preventive therapy based on IGRA results
Predictive value of IGRA: HIV-negative
subjects
1414 contacts followed in Hamburg, Germany
Diel et al, AJRCCM 2010
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 TSPOT.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-house32 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

Sensitivity and specificity

Predictive value of IGRA for active TB development

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
Luetkeme
yer,
AJRCCM
2007
Clark,
Clin Exp
Imm
2007
Karam,
Plos ONE
2008
Rabi,
Plos ONE
2008
Test
QFT
ELISPOT
QFT
QFT
ELISPOT
QFT
N.
Patients
590
111
196
201
247
84
4 (24%)
12 (57%)
5 (16%)
4 (6%)
6 (16%)
6 (46%)
100-200
1 (3%)
4 (19%)
4 (3.6%)
12 (31%)
3 (15%)
201-300
5 (8%)
3 (14%)
10 (26)
3 (13%)
CD4
home-made
home-made
per ml
<100
1 (NA)
IGRAs in subjects with for autoimmune
diseases under immune suppressive
therapy
Solovic et al, ERJ 2010
RD1-IGRA
BCG-vaccination
NTM
Positive
RD1-IGRA
Positive
Active TB disease
M. tuberculosis
infection/disease
Latent TB infection:
Recently acquired or
•
•
Remotely acquired
Positive RD1-IGRA do not distinguish
active TB disease and LTBI
Need of…

Standardized test (laboratory test)

M. tuberculosis-specific reagents

Possibility to discriminate between the different
stages of tuberculosis
Agenda

LTBI definition

TST

IGRA

New experimental tests
Why is it important to distinguish between
latent infection and active TB disease?

To provide a correct diagnosis:

Active TB disease:




Latent infection
To provide a correct and efficacious therapy:



Organ destruction and/or death
Spread of infection in the community
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
New experimental tests




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
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-γ response to RD1 selected peptides
is associated to active TB
Modified Vincenti
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
Likely
Remote LTBI
Recent Infection
Higher need of
chemoprophilaxis
with
active
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+
Likely
Active TB
Likely
No active TB:
Recent Infection,
Remote Infection,
past cured TB
New experimental tests




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)
Detection of IP-10 in the plasma from
QuantiFERON-TB Gold In-tube
INCREASE OF SENSITIVITY !
From Ruwald et al, modified Microbes Infection 2007
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
New experimental tests




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, 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
New experimental tests




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, 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

New experimental tests
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 University
Cleveland, 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