ORIGINAL ARTICLE
Department of Paediatrics and 7Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria,
Australia; 2Infectious Diseases Unit and 5Clinical Epidemiology and Biostatistics Unit, Royal Childrens Hospital Melbourne, Parkville,
Victoria, Australia; 3Murdoch Childrens Research Institute, Parkville, Victoria, Australia; 4Academic Unit of Clinical and Experimental
Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom; 6Infectious Diseases Unit, University
Childrens Hospital Basel, Basel, Switzerland; 8Institute of Infectious Diseases and Molecular Medicine and School of Child
and Adolescent Health, University of Cape Town, Cape Town, South Africa; and 9Centre for International Child Health,
Parkville, Victoria, Australia
Abstract
Rationale: Current immunodiagnostic tests for tuberculosis
(TB), including the tuberculin skin test and IFN-g release assay
(IGRA), have signicant limitations, which include their inability
to distinguish between latent TB infection (LTBI) and active
TB, a distinction critical for clinical management.
Objectives: To identify mycobacteria-specic cytokine
( Received in original form January 11, 2015; accepted in final form May 29, 2015 )
Supported by the Australian National Health and Medical Research Council (Project Grant No. 1011200), the John Burge Trust, the Myer Foundation, the
Aranday Foundation, and the Murdoch Childrens Research Institute. M.T. and N.R. were supported by Fellowship awards from the European Society for
Paediatric Infectious Diseases and by International Research Scholarship awards from The University of Melbourne.
Author Contributions: Conception and design, M.T., B.D., S.D., N.R., R.R.-B., S.M.G., T.C., and N.C. Acquisition of data, M.T., B.D., B.F., and K.C.-B.
Analysis and interpretation of data, M.T., B.D., S.D., N.R., B.F., K.C.-B., V.C., C.Z., R.R.-B., W.H., S.M.G., T.C., and N.C. Drafting the manuscript, M.T. and
N.C. Revising the manuscript for important intellectual content, B.D., S.D., N.R., B.F., K.C.-B., V.C., C.Z., R.R.-B., W.H., S.M.G., and T.C.
Correspondence and requests for reprints should be addressed to Nigel Curtis, Ph.D., Department of Paediatrics, The University of Melbourne, The Royal
Childrens Hospital, Flemington Road, Parkville, VIC 3052, Australia. E-mail: nigel.curtis@rch.org.au.
This article has an online supplement, which is accessible from this issues table of contents at www.atsjournals.org
Am J Respir Crit Care Med Vol 192, Iss 4, pp 485499, Aug 15, 2015
Copyright 2015 by the American Thoracic Society
Originally Published in Press as DOI: 10.1164/rccm.201501-0059OC on June 1, 2015
Internet address: www.atsjournals.org
485
ORIGINAL ARTICLE
At a Glance Commentary
Scientic Knowledge on the
Subject: Current immunodiagnostic
mycobacteria-specic cytokine
responses that allow the distinction
between TB-infected and TBuninfected individuals, as well as
between LTBI and active TB. Several
of the cytokine biomarkers showed
better performance characteristics
than IFN-g, which forms the basis
of IFN-g release assays. Addition
of these biomarkers into future
immunodiagnostic assays for TB is
likely to result in higher assay
sensitivity while retaining high
specicity, and it could potentially
allow the distinction between LTBI and
active TB based on a blood test alone.
Approximately one-third of the worlds
population is infected with Mycobacterium
tuberculosis (MTB), and there are more
than 8 million new cases of active
tuberculosis (TB) (i.e., TB disease) resulting
in an estimated 1.3 million deaths each
year (1, 2). Children account for an
increasing proportion of TB cases, in
both high-resource and low-resource
settings (3).
Despite advances in diagnostics,
microbiologic conrmation of TB in
childhood remains challenging, primarily
because most children have paucibacillary
disease, reducing the yield of conventional
microbiologic methods. Newer molecular
diagnostic techniques, including the Xpert
MTB/RIF assay, show promise, but also
perform considerably less well in patients
with paucibacillary disease (4).
Existing immunodiagnostic tests for
TB also have considerable limitations (5, 6).
The tuberculin skin test (TST) is
operator-dependent and has limited
specicity. False-positive results can
486
Methods
Participants
American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015
ORIGINAL ARTICLE
volar surface of the lower arm, and any
resulting induration was recorded after 4872
hours. In addition, blood was obtained for
the QFT In-Tube (QFT-GIT) assay
(Cellestis/Qiagen, Carnegie, Australia), and
an additional 10 ml for whole-blood assays
were collected in heparinized tubes. The
QFT-GIT was processed and interpreted at
the Victorian Infectious Diseases Reference
Laboratories in accordance with the
manufacturers instructions. Polymerase
chain reaction testing for MTB, for which
a Taqman real-time polymerase chain
reaction (Applied Biosystems, Waltham,
MA) targeting the insertion sequence IS6110
was used, was also performed at the
Victorian Infectious Diseases Reference
Laboratories using previously described
methods (20).
Categorization of Participants and
Denitions
0
14
59
>10
>10
*
,10
Negative
Negative
Negative
Negative
Positive
*
Positive
Definition of abbreviations: IGRA = IFN-g release assay; LTBI = latent TB infection; QFT-GIT =
QuantiFERON-TB Gold In-Tube assay; TB = tuberculosis; TST = tuberculin skin test.
*Microbiologic confirmation or presence of two of three diagnostic criteria in conjunction with
response to antituberculous treatment (irrespective of TST and IGRA result; see METHODS section).
Results
A total of 149 patients were recruited.
Nine participants were excluded: three
did not return for TST reading, insufcient
blood was obtained in three, a laboratory
error occurred during the QFT-GIT
487
ORIGINAL ARTICLE
processing in one, and the QFT-GIT result
was indeterminate in two (both failed
positive controls). Therefore, a total of
140 patients were included in the analysis.
The six participants with active
TB comprised three patients with
microbiologically conrmed TB (one case
with intrathoracic TB; two cases with lymph
node TB), and three patients without
microbiologic conrmation who fullled
the study criteria for active TB (two cases
with pulmonary TB; one case with spinal
TB). All six patients had both a positive
TST (range, 1325 mm induration) and
a positive QFT-GIT result, and resolution
of symptoms with antituberculous therapy.
Additional patient data are shown in
Table E1 in the online supplement.
Table 2 shows the demographic and
other details of the participants in the four
major diagnostic categories (uninfected,
common discordance, LTBI, and active
TB). In the remaining three diagnostic
categories there were six probable
uninfected, ve possible discordance,
and four reverse discordance cases.
Mycobacteria-Specic Cytokine
Responses in Supernatants
Table 2. Demographic and Other Details of Study Participants in Each Diagnostic Category
Total Cohort
(n = 140)
Uninfected
(n = 75)
Common Discordance
(n = 28)
LTBI
(n = 16)
Active TB
(n = 6)
8.3 (3.712.8)
6.3 (2.810.9)
12.1 (6.014.5)
11.6 (6.014.2)
15.0 (12.116.2)
60
53
9
18
(42.8)
(37.9)
(6.4)
(12.9)
31
24
5
15
41
29
9
59
2
81
8.0
(29.3)
(20.7)
(6.4)
(42.1)
(1.4)
(57.9)
(3.536.0)
14
10
4
47
(41.3)
(32.0)
(6.7)
(20)
(18.7)
(13.3)
(5.3)
(62.7)
0
28 (37.3)
5.5 (2.033.0)
9
13
4
2
8
7
4
8
1
20
17.0
(32.1)
(46.4)
(14.3)
(7.1)
10 (62.5)
5 (31.3)
0
1 (6.3)
(28.6)
(25.0)
(14.3)
(28.6)
(3.6)
(71.4)
(6.036.0)
10 (62.5)
5 (31.3)
0
1 (6.3)
0
15 (93.8)
7.0 (4.036.0)
5 (83.3)
1 (16.7)
0
0
5 (83.3)
1 (16.7)
0
0
0
6 (100)
21.0 (3.069.0)
75 (53.6)
58 (42.4)
7 (5.0)
23 (30.7)
49 (65.3)
3 (4.0)
21 (75.0)
4 (14.2)
3 (10.7)
14 (87.5)
1 (6.3)
1 (6.3)
5 (83.3)
1 (16.7)
0
67 (47.9)
73 (52.1)
22 (29.3)
53 (70.7)
18 (64.3)
10 (35.7)
12 (75.0)
4 (25.0)
5 (83.3)
1 (16.7)
89 (63.6)
51 (36.4)
54 (72.0)
21 (28.0)
14 (50.0)
14 (50.0)
8 (50.0)
8 (50.0)
1 (16.7)
5 (83.3)
34 (24.3)
33 (23.6)
22 (15.7)
14 (18.6)
23 (30.7)
17 (22.7)
9 (32.1)
3 (10.7)
2 (7.1)
4 (25.0)
3 (18.8)
1 (6.3)
1 (16.7)
0
0
488
American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015
ORIGINAL ARTICLE
A
IFN-
IP-10
TNF-
p < 0.0001
p < 0.0001
p < 0.0001
600000
2000
500000
1600
450000
1400
400000
1200
350000
1000
300000
800
250000
600
200000
400
150000
200
100000
50000
200
300
400
100
300
200
200
300
400
600
800
1000
100
IL-10
IL-12(p40)
IL-13
p = 0.0061
p < 0.0001
1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
0
100
200
300
400
500
IL-15
50
40
40
30
30
10
50
30
10
10
20
20
10
30
40
30
40
50
100
200
300
400
5000
5000
4000
3000
2000
5000
1000
0
100
20
MCP-3
p = 0.1038
p = 0.1701
10000
200
20
70
MCP-1
p < 0.0001
300
50
50
1000
1200
GM-CSF
p = 0.1647
20
10
10
10
30
40
30
20
600
400
200
70
20
IL-17
p = 0.4083
40
10000
15000
1000
20000
2000
25000
3000
30000
40000
60000
80000
4000
40
0
60
10
80
20
50
100
100
100
200
300
100
200
400
600
5000
RANTES
MIP-1
p < 0.0001
p = 0.3249
70000
50000
30000
4000
10000
3000
10000
2000
30000
1000
50000
70000
3000
p = 0.1041
100
2000
500
100
IL-8
200
1000
600
200
p = 0.7564
300
5000
1200
1000
800
700
400
IL-6
400
13000
11000
9000
7000
6000
IL-2
p < 0.0001
p = 0.0642
500
concentration (pg/ml)
1600
1500
650
600
550
500
450
400
350
300
250
200
150
100
50
0
50
550000
1800
IL-1ra
p < 0.0001
500
90000
110000
130000
Figure 1. Box plot with Tukey whiskers showing background-corrected cytokine concentrations in uninfected participants (white) and participants with
common discordance (yellow), latent tuberculosis infection (orange), and active tuberculosis (red) in ESAT-6 (early secretory antigenic target-6) (A),
CFP-10 (10-kD culture filtrate protein) (B), and PPD- (C) stimulated samples. Statistical differences were analyzed using Kruskal-Wallis tests; the
corresponding P values are shown at the top of each graph. The results of additional two-group comparisons are detailed in Table 3. GM-CSF =
granulocyte-macrophage colonystimulating factor; IP-10 = interferon-inducible protein-10; MCP = monocyte chemotactic protein; MIP-1b = macrophage
inflammatory protein 1b; RANTES = regulated upon activation, normal T-cell expressed and secreted; TNF-a = tumor necrosis factor a.
489
ORIGINAL ARTICLE
IFN-
IP-10
TNF-
IL-1ra
IL-2
p < 0.0001
p < 0.0001
p < 0.0001
p < 0.0001
p < 0.0001
B
4000
3000
2000
1000
600
1600000
1200000
800000
400000
400000
500
350000
400
300000
800
600
400
200
150
800
600
400
200
2000
1500
1000
500
400
100
350
300
250
250000
300
100
100
200000
200
200
150000
100
100000
50
100
300
50000
100
200
50000
50
400
500
1000
1500
0
50
IL-6
IL-8
IL-10
IL-12(p40)
IL-13
p = 0.1354
p = 0.8776
p = 0.0367
p = 0.1895
p < 0.0001
400
5000
300
200
100
40
50
30
40
20
30
5000
10000
30
10
200
40
10
100
1000
800
600
400
200
50
20
10
10
20
20
10
20
300
concentration (pg/ml)
150
200
30
400
30
15000
40
600
2000
3000
50
200
400
600
20000
50000
60000
40
500
10
20
40
60
50
60
IL-15
IL-17
GM-CSF
MCP-1
MCP-3
p = 0.9146
p = 0.4539
p = 0.0013
p = 0.1643
p = 0.0155
40
50
300
40
30
30
200
20
20
100
0
0
10
0
20
200
400
600
800
50
100
200
300
1000
0
40000
1000
50000
2000
60000
3000
4000
80000
90000
100000
5000
5000
10000
15000
RANTES
MIP-1
p < 0.0001
p = 0.1390
25000
20000
5000
30000
4000
10000
3000
30000
70000
40
20
40
60
2000
10000
100
30
10
3000
20000
10
10
10000
20000
0
10000
2000
20000
1000
30000
40000
1000
50000
60000
2000
3000
70000
80000
4000
Figure 1. (Continued).
American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015
ORIGINAL ARTICLE
C
IFN-
IP-10
TNF-
IL-1ra
IL-2
p < 0.0001
p < 0.0001
p < 0.0001
p < 0.0040
p < 0.0001
6000
700000
50000
40000
25000
600000
20000
5000
3000
2000
2000
4500
5000
4000
500000
3500
1500
4000
3000
400000
15000
3000
2500
1000
300000
2000
10000
2000
200000
1500
500
5000
100000
1000
1000
500
0
0
IL-6
IL-8
IL-10
IL-12(p40)
IL-13
p < 0.0001
p = 0.0719
p = 0.0007
p < 0.0001
p = 0.0003
35000
800
250000
30000
200000
5000
4000
3000
2000
700
600
300
700
600
250
500
200
400
150
300
100
25000
150000
20000
15000
100000
10000
200
500
50
50000
concentration (pg\ml)
1000
100
5000
50
IL-15
IL-17
GM-CSF
MCP-1
MCP-3
p = 0.2734
p = 0.3771
p < 0.0001
p = 0.4549
p = 0.2129
300
200
100
40
35
30
3000
300000
2000
250000
40000
35000
30000
50
25
1500
200000
25000
20
15
1000
150000
20000
15000
10
100000
500
5
50
10000
50000
5000
0
0
10
100
MIP-1
5000
RANTES
p < 0.0001
p = 0.3126
45000
80000
40000
60000
35000
500
40000
30000
20000
25000
20000
15000
20000
10000
40000
5000
60000
0
80000
Figure 1. (Continued).
ORIGINAL ARTICLE
Table 3. Comparisons of Median Cytokine Responses (P Values) from the Data Shown in Figure 1
Stimulant
ESAT-6
CFP-10
PPD
Cytokine
Kruskal-Wallis
P Value
Uninfected
vs. CD
Uninfected
vs. LTBI
Uninfected vs.
Active TB
CD vs.
LTBI
CD vs.
Active TB
LTBI vs.
Active TB
IFN-g
IP-10
TNF-a
IL-1ra
IL-2
IL-6
IL-8
IL-10
IL-12(p40)
IL-13
IL-15
IL-17
GM-CSF
MCP-1
MCP-3
MIP-1b
RANTES
IFN-g
IP-10
TNF-a
IL-1ra
IL-2
IL-6
IL-8
IL-10
IL-12(p40)
IL-13
IL-15
IL-17
GM-CSF
MCP-1
MCP-3
MIP-1b
RANTES
IFN-g
IP-10
TNF-a
IL-1ra
IL-2
IL-6
IL-8
IL-10
IL-12(p40)
IL-13
IL-15
IL-17
GM-CSF
MCP-1
MCP-3
MIP-1b
RANTES
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.0642
0.7564
0.1042
0.0061
<0.0001
0.4083
0.1647
<0.0001
0.1701
0.1038
<0.0001
0.3249
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.1354
0.8776
0.0367
0.1895
<0.0001
0.9146
0.4539
0.0013
0.1643
0.0155
<0.0001
0.1390
<0.0001
<0.0001
<0.0001
0.0040
<0.0001
<0.0001
0.0719
0.0007
<0.0001
0.0003
0.2734
0.3771
<0.0001
0.4549
0.2129
<0.0001
0.3126
0.0147
0.0026
0.0504
0.2925
<0.0001*
0.0080
0.0269
0.6012
0.1883
0.1588
0.2630
0.2908
0.4653
0.0202
0.0279
0.0556
0.4064
0.0149
0.4630
<0.0001*
<0.0001*
<0.0001*
0.0154
<0.0001*
<0.0001*
0.3737
<0.0001*
0.0007*
<0.0001*
0.0004*
<0.0001*
<0.0001*
<0.0001*
0.0005*
<0.0001*
0.0255
<0.0001*
<0.0001*
<0.0001*
<0.0001*
<0.0001*
<0.0001*
0.0013*
<0.0001*
0.0250
0.0066
0.0012*
0.0120
0.0041
<0.0001*
<0.0001*
<0.0001*
0.6843
<0.0001*
<0.0001*
0.0022
0.0002*
0.0061
<0.0001*
0.0002*
<0.0001*
<0.0001*
<0.0001*
<0.0001*
<0.0001*
0.0540
<0.0001*
0.0058
<0.0001*
<0.0001*
<0.0001*
<0.0001*
0.0003*
<0.0001*
0.2635
<0.0001*
0.0104
0.2948
<0.0001*
<0.0001*
0.0007*
<0.0001*
0.0044
<0.0001*
0.0001*
0.0058
<0.0001*
0.0173
0.0002*
0.0024*
0.0001*
<0.0001*
<0.0001*
0.0180
<0.0001*
0.6258
0.0127
<0.0001*
0.0005*
0.0128
0.0013*
0.0029
0.0338
0.0007*
0.7325
0.1368
0.0192
0.5310
0.0404
0.0832
0.2134
0.0180
0.0570
0.0043
0.5747
0.0790
0.3602
0.8073
0.4068
0.3667
0.0008*
0.0007*
0.0004*
0.0007*
0.0010*
0.2433
0.0006*
0.0129
0.0006*
0.0011*
0.0018
0.0005*
0.0025
0.0021
0.8037
0.0004*
0.0377
1
0.0018
0.0025
0.5877
0.0016*
0.3203
0.0025
0.0100
0.0239
0.0377
0.8922
0.1245
0.3907
0.3020
0.8828
0.0183
0.0183
0.5070
0.3451
0.0650
0.7124
0.0900
0.0900
0.6058
0.0121
0.1048
0.4174
0.9412
0.0765
0.7681
0.9105
0.1404
0.1845
0.0270
0.0553
0.0032
0.4610
0.0122
0.0004*
0.5070
0.3763
0.4610
0.7681
Definition of abbreviations: CD = common discordance (tuberculin skin test1/IFN-g release assay2); CFP-10 = 10-kD culture filtrate protein; ESAT-6 =
early secretory antigenic target-6; GM-CSF = granulocyte-macrophage colonystimulating factor; IP-10 = interferon-inducible protein-10; LTBI = latent TB
infection; MCP = monocyte chemotactic protein; MIP-1b = macrophage inflammatory protein 1b; RANTES = regulated upon activation, normal T-cell
expressed and secreted; TB = tuberculosis; TNF-a = tumor necrosis factor a.
Statistically significant P values are shown in bold.
*Significant at Bonferroni-corrected significance level of 0.0016.
American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015
10.2
0.8
659.4
90.9
81.8
95.5
97.3
89.3
97.3
10,820.0
2,204.0
4,6571.0
Cut-off
TNF-a
IL-1ra
IL-2
IL-13
MIP-1b
95.5
86.4
95.5
96.0
93.3
81.3
5.99
1.9
125.3
95.5
86.4
95.5
88.0
85.3
84.0
228.5
223.6
279.7
81.8
77.3
50.0
69.3
78.7
46.7
16.7
4.7
383.7
95.5
90.9
100.0
97.3
94.7
96.0
0.32
0.05
103.5
77.3
63.6
77.3
89.3
86.7
69.3
158.6
50.2
9618.0
90.9
77.3
81.8
70.7
68.0
60.0
Sens. Spec.
Sens. Spec.
Sens. Spec.
Sens. Spec.
Sens. Spec.
Sens. Spec.
(%)
(%) Cut-off (%)
(%)
Cut-off (%)
(%)
Cut-off (%)
(%)
Cut-off (%)
(%)
Cut- off (%)
(%)
IP-10
Definition of abbreviations: CFP-10 = 10-kD culture filtrate protein; ESAT-6 = early secretory antigenic target-6; IP-10 = interferon-inducible protein-10; MIP-1b = macrophage inflammatory
protein 1b; Sens. = sensitivity; Spec. = specificity; TB = tuberculosis; TNF-a = tumor necrosis factor a.
All cut-offs are in pg/ml. Bold numbers indicate that both sensitivity and specificity exceed 80%.
ESAT-6
CFP-10
PPD
Sens. Spec.
Cut-off (%)
(%)
IFN-g
Table 4. Results of the Receiver Operating Characteristic Analyses of the Seven Mycobacteria-Specic Cytokine Responses with the Potential Ability to
Discriminate between TB-uninfected and TB-infected Individuals
ORIGINAL ARTICLE
493
ORIGINAL ARTICLE
CFP-10
100
80
80
80
60
40
Sensitivity (%)
100
20
60
40
20
AUC=0.937
0
0
20
40
60
80 100
60
80
40
Sensitivity (%)
80
60
60
40
20
AUC=0.961
40
60
20
40
60
80 100
80
Sensitivity (%)
80
Sensitivity (%)
80
60
40
20
AUC=0.944
0
20
40
60
20
40
60
80
Sensitivity (%)
80
Sensitivity (%)
80
60
40
20
AUC=0.836
0
20
40
60
40
60
80 100
60
40
20
AUC=0.809
80 100
20
AUC=0.959
80 100
100
20
80 100
40
100
40
60
60
60
40
20
AUC=0.896
80 100
20
AUC=0.944
100
20
80 100
40
100
40
60
60
60
40
20
AUC=0.898
80 100
20
80
20
AUC=0.987
80 100
100
Sensitivity (%)
IP-10
Sensitivity (%)
40
100
TNF-
20
100
Sensitivity (%)
40
20
IL-1ra
60
20
AUC=0.876
Sensitivity (%)
PPD
100
Sensitivity (%)
IFN-
Sensitivity (%)
ESAT-6
20
40
60
80 100
AUC=0.533
0
0
20
40
60
80 100
Figure 2. Receiver operating characteristic curves for IFN-g, interferon-inducible protein-10 (IP-10), tumor
necrosis factor a (TNF-a), IL-1ra, IL-2, IL-13, and macrophage inflammatory protein-1b (MIP-1b)
responses according to antigenic stimulant (early secretory antigenic target-6 [ESAT-6], 10-kD culture
filtrate protein [CFP-10], or PPD). Case values comprised background-corrected cytokine concentrations in
participants with latent tuberculosis infection or active tuberculosis (tuberculosis-infected group); control
values comprised cytokine concentrations in uninfected participants. AUC = area under the curve.
Discussion
Our data show that in addition to IFN-g,
which forms the basis of existing
American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015
ORIGINAL ARTICLE
495
CFP-10
100
80
80
80
60
40
Sensitivity (%)
100
20
60
40
20
40
60
AUC=0.941
80 100
40
20
40
60
100
80
80
80
40
20
Sensitivity (%)
100
60
40
20
AUC=0.859
0
0
20
40
60
AUC=0.790
0
20
40
60
Sensitivity (%)
80
Sensitivity (%)
80
60
40
20
0
0
20
40
60
80 100
20
40
60
80 100
80
AUC=0.900
AUC=0.739
80 100
100
20
80 100
40
100
40
60
60
100
60
40
20
80 100
20
100
60
AUC=0.998
80 100
Sensitivity (%)
IL-13
Sensitivity (%)
MIP-1
60
20
20
AUC=0.958
Sensitivity (%)
PPD
100
Sensitivity (%)
IL-2
Sensitivity (%)
ESAT-6
60
40
20
AUC=0.800
0
0
20
40
60
80 100
AUC=0.822
0
0
20
40
60
80 100
Figure 2. (Continued.)
ORIGINAL ARTICLE
ESAT-6
CFP-10
p=0.0183
PPD
p=0.0121
2000
1000
1000
p=0.0553
8000
800
700
200
7000
6000
800
150
TNF-
5000
600
4000
100
3000
400
2000
50
200
1000
0
I
TB
I
TB
TB
iv
ct
LT
iv
ct
p=0.0183
TB
p=0.0032
p=0.1048
200
tiv
Ac
400
1200
300
1000
100
200
IL-1ra
concentration (pg/ml)
BI
TB
800
100
600
0
0
400
100
100
200
200
0
200
700
800
300
500
600
I
TB
200
I
TB
TB
iv
ct
BI
LT
iv
ct
p=0.9412
10
600
500
400
300
10
10
200
15
15
100
20
B
LT
iv
t
Ac
TB
TB
p=0.0004
10
20
tiv
Ac
p=0.9119
IL-10
TB
B
LT
iv
t
Ac
TB
BI
LT
TB
iv
t
Ac
Figure 3. Background-corrected tumor necrosis factor (TNF)-a, IL-1ra, and IL-10 concentrations
in participants with latent tuberculosis infection (LTBI) (circles) and active tuberculosis (TB)
(squares) in early secretory antigenic target-6 (ESAT-6), 10-kD culture filtrate protein (CFP-10),
and PPD-stimulated samples. The bars indicate the median concentrations. The dotted lines
indicate the optimal cut-offs for the distinction between participants with LTBI and those with active
TB. Statistical differences were analyzed using Mann-Whitney U tests; the corresponding P values
are shown above each bracket.
496
American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015
ORIGINAL ARTICLE
A
B
1400
1200
1000
800
600
400
200
1000
200
800
Active TB
700
LTBI
Uninfected
600
500
400
300
200
100
1000
2000
3000
4000
5000
6000
1000
100
1000
2000
3000
4000
5000
6000
Figure 4. Background-corrected tumor necrosis factor (TNF)-a, IL-1ra, and IL-10 concentrations
in uninfected participants (gray diamonds), participants with latent tuberculosis infection (LTBI) (orange
circles), and active tuberculosis (TB) (red squares) in PPD-stimulated samples. (A) Combination of TNF-a/IL1ra. (B) Combination of TNF-a/IL-10. The dotted lines indicate the optimal cut-offs for the distinction between
participants with LTBI and those with active TB (TNF-a, 800 pg/ml; IL-1ra, 450 pg/ml; IL-10, 100 pg/ml).
References
1. Dye C. Global epidemiology of tuberculosis. Lancet 2006;367:938940.
2. World Health Organization. Global tuberculosis report 2013 [accessed
2015 Jul 22]. Available from: http://www.who.int/tb/publications/
global_report/en/
3. Marais BJ, Gie RP, Schaaf HS, Beyers N, Donald PR, Starke JR.
Childhood pulmonary tuberculosis: old wisdom and new challenges.
Am J Respir Crit Care Med 2006;173:10781090.
4. Nicol MP, Workman L, Isaacs W, Munro J, Black F, Eley B, Boehme CC,
Zemanay W, Zar HJ. Accuracy of the Xpert MTB/RIF test for the
diagnosis of pulmonary tuberculosis in children admitted to hospital in
Cape Town, South Africa: a descriptive study. Lancet Infect Dis
2011;11:819824.
5. Tebruegge M, Connell T, Curtis N. Tuberculosis in children. N Engl J
Med 2012;367:1568.
6. Tebruegge M, Ritz N, Curtis N, Shingadia D. Diagnostic tests for
childhood tuberculosis past imperfect, present tense and future
perfect? Pediatr Infect Dis J [online ahead of print] 22 Jun 2015; DOI:
10.1097/INF.0000000000000796.
497
ORIGINAL ARTICLE
13. Haustein T, Ridout DA, Hartley JC, Thaker U, Shingadia D, Klein NJ,
Novelli V, Dixon GL. The likelihood of an indeterminate test result from
a whole-blood interferon-gamma release assay for the diagnosis of
Mycobacterium tuberculosis infection in children correlates with age
and immune status. Pediatr Infect Dis J 2009;28:669673.
14. Tebruegge M, de Graaf H, Sukhtankar P, Elkington P, Marshall B,
Schuster H, Patel S, Faust SN. Extremes of age are associated with
indeterminate QuantiFERON-TB gold assay results. J Clin Microbiol
2014;52:26942697.
15. Kaufmann SH. How can immunology contribute to the control of
tuberculosis? Nat Rev Immunol 2001;1:2030.
16. Ruhwald M, Ravn P. Biomarkers of latent TB infection. Expert Rev
Respir Med 2009;3:387401.
17. Clifford V, Zufferey C, Street A, Denholm J, Tebruegge M, Curtis N.
Cytokine biomarkers for monitoring anti-tuberculous therapy:
a systematic review. Tuberculosis (Edinb) 2015;95:217228.
18. Naseer A, Naqvi S, Kampmann B. Evidence for boosting
Mycobacterium tuberculosis-specic IFN-gamma responses at 6
weeks following tuberculin skin testing. Eur Respir J 2007;29:
12821283.
19. Ritz N, Yau C, Connell TG, Tebruegge M, Leslie D, Curtis N. Absence of
interferon-gamma release assay conversion following tuberculin skin
testing. Int J Tuberc Lung Dis 2011;15:767769.
20. Globan M, Fyfe J. Mycobacterium tuberculosis complex. In: Schuller M,
Sloots TP, James GS, Halliday CL, Carter IWJ, editors. PCR for clinical
microbiology, 1st ed. New York, NY: Springer; 2010. pp. 165170.
21. American Thoracic Society and the Centers for Disease Control and
Prevention. Diagnostic standards and classication of tuberculosis in
adults and children. Ofcial statement of the American Thoracic
Society and the Centers for Disease Control and Prevention. Am J
Respir Crit Care Med 2000;161:13761395.
22. Whiting P, Rutjes AW, Reitsma JB, Bossuyt PM, Kleijnen J. The
development of QUADAS: a tool for the quality assessment of
studies of diagnostic accuracy included in systematic reviews. BMC
Med Res Methodol 2003;3:25.
23. Harari A, Rozot V, Bellutti Enders F, Perreau M, Stalder JM, Nicod LP,
Cavassini M, Calandra T, Blanchet CL, Jaton K, et al. Dominant TNFa1 Mycobacterium tuberculosis-specic CD41 T cell responses
discriminate between latent infection and active disease. Nat Med
2011;17:372376.
24. Alsleben N, Ruhwald M, Russmann
Khourouj V, Pedron
B,
Jeljeli M, Gressens P, Faye A, Sterkers G. Cytokine responses to
quantiferon peptides in pediatric tuberculosis: a pilot study. J Infect
2014;68:6270.
26. Frahm M, Goswami ND, Owzar K, Hecker E, Mosher A, Cadogan E,
Nahid P, Ferrari G, Stout JE. Discriminating between latent and
active tuberculosis with multiple biomarker responses. Tuberculosis
(Edinb) 2011;91:250256.
27. Whittaker E, Gordon A, Kampmann B. Is IP-10 a better biomarker for
active and latent tuberculosis in children than IFNgamma? PLoS One
2008;3:e3901.
28. Yassin MA, Petrucci R, Garie KT, Harper G, Arbide I, Aschalew M,
Merid Y, Kebede Z, Bawazir AA, Abuamer NM, et al. Can interferongamma or interferon-gamma-induced-protein-10 differentiate
tuberculosis infection and disease in children of high endemic areas?
PLoS One 2011;6:e23733.
29. Ruhwald M, Bjerregaard-Andersen M, Rabna P, Eugen-Olsen J, Ravn
P. IP-10, MCP-1, MCP-2, MCP-3, and IL-1RA hold promise as
biomarkers for infection with M. tuberculosis in a whole blood based
T-cell assay. BMC Res Notes 2009;2:19.
30. Ruhwald M, Bjerregaard-Andersen M, Rabna P, Kofoed K, EugenOlsen J, Ravn P. CXCL10/IP-10 release is induced by incubation of
whole blood from tuberculosis patients with ESAT-6, CFP10 and
TB7.7. Microbes Infect 2007;9:806812.
31. Gasperini S, Marchi M, Calzetti F, Laudanna C, Vicentini L, Olsen H,
Murphy M, Liao F, Farber J, Cassatella MA. Gene expression and
production of the monokine induced by IFN-gamma (MIG), IFN-inducible
498
T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein10 (IP-10) chemokines by human neutrophils. J Immunol 1999;162:
49284937.
32. Colizzi V. In vivo and in vitro administration of interleukin 2-containing
preparation reverses T-cell unresponsiveness in Mycobacterium
bovis BCG-infected mice. Infect Immun 1984;45:2528.
33. Hubbard RD, Collins FM. Immunomodulation of mouse macrophage
killing of Mycobacterium avium in vitro. Infect Immun 1991;59:
570574.
34. Biselli R, Mariotti S, Sargentini V, Sauzullo I, Lastilla M,
Mengoni F, Vanini V, Girardi E, Goletti D, D Amelio R, et al.
Detection of interleukin-2 in addition to interferon-gamma
discriminates active tuberculosis patients, latently infected
individuals, and controls. Clin Microbiol Infect 2010;16:
12821284.
35. Casey R, Blumenkrantz D, Millington K, Montamat-Sicotte D, Kon OM,
Wickremasinghe M, Bremang S, Magtoto M, Sridhar S, Connell D,
et al. Enumeration of functional T-cell subsets by uorescenceimmunospot denes signatures of pathogen burden in tuberculosis.
PLoS One 2010;5:e15619.
36. Chiappini E, Della Bella C, Bonsignori F, Sollai S, Amedei A, Galli L,
Niccolai E, Del Prete G, Singh M, DElios MM, et al. Potential role of
M. tuberculosis specic IFN-g and IL-2 ELISPOT assays in
discriminating children with active or latent tuberculosis. PLoS One
2012;7:e46041.
37. Krummel B, Strassburg A, Ernst M, Reiling N, Eker B, Rath H, Hoerster
R, Wappler W, Glaewe A, Schoellhorn V, et al. Potential role for IL-2
ELISpot in differentiating recent and remote infection in tuberculosis
contact tracing. PLoS One 2010;5:e11670.
38. Lighter-Fisher J, Peng CH, Tse DB. Cytokine responses to
QuantiFERON peptides, puried protein derivative and recombinant
ESAT-6 in children with tuberculosis. Int J Tuberc Lung Dis 2010;14:
15481555.
39. Wang S, Diao N, Lu C, Wu J, Gao Y, Chen J, Zhou Z, Huang H, Shao L,
Jin J, et al. Evaluation of the diagnostic potential of IP-10 and IL-2 as
biomarkers for the diagnosis of active and latent tuberculosis in
a BCG-vaccinated population. PLoS One 2012;7:e51338.
40. Chegou NN, Detjen AK, Thiart L, Walters E, Mandalakas AM, Hesseling
AC, Walzl G. Utility of host markers detected in Quantiferon
supernatants for the diagnosis of tuberculosis in children in a highburden setting. PLoS One 2013;8:e64226.
41. Yu Y, Zhang Y, Hu S, Jin D, Chen X, Jin Q, Liu H. Different patterns of
cytokines and chemokines combined with IFN-g production reect
Mycobacterium tuberculosis infection and disease. PLoS One 2012;
7:e44944.
42. Sutherland JS, de Jong BC, Jeffries DJ, Adetifa IM, Ota MO. Production
of TNF-alpha, IL-12(p40) and IL-17 can discriminate between active
TB disease and latent infection in a West African cohort. PLoS One
2010;5:e12365.
43. Farhat M, Greenaway C, Pai M, Menzies D. False-positive
tuberculin skin tests: what is the absolute effect of BCG and nontuberculous mycobacteria? Int J Tuberc Lung Dis 2006;10:
11921204.
44. Connell TG, Ritz N, Paxton GA, Buttery JP, Curtis N, Ranganathan
SC. A three-way comparison of tuberculin skin testing,
QuantiFERON-TB gold and T-SPOT.TB in children. PLoS One
2008;3:e2624.
45. Connell TG, Curtis N, Ranganathan SC, Buttery JP. Performance of
a whole blood interferon gamma assay for detecting latent infection
with Mycobacterium tuberculosis in children. Thorax 2006;61:
616620.
46. Hill PC, Brookes RH, Adetifa IM, Fox A, Jackson-Sillah D, Lugos MD,
Donkor SA, Marshall RJ, Howie SR, Corrah T, et al. Comparison of
enzyme-linked immunospot assay and tuberculin skin test in healthy
children exposed to Mycobacterium tuberculosis. Pediatrics 2006;
117:15421548.
47. Wallis RS. Infectious complications of tumor necrosis factor blockade.
Curr Opin Infect Dis 2009;22:403409.
48. Singh JA, Wells GA, Christensen R, Tanjong Ghogomu E, Maxwell L,
Macdonald JK, Filippini G, Skoetz N, Francis D, Lopes LC, et al.
Adverse effects of biologics: a network meta-analysis and Cochrane
overview. Cochrane Database Syst Rev 2011;2:CD008794.
American Journal of Respiratory and Critical Care Medicine Volume 192 Number 4 | August 15 2015
ORIGINAL ARTICLE
49. Ruth JH, Bienkowski M, Warmington KS, Lincoln PM, Kunkel SL, Chensue
SW. IL-1 receptor antagonist (IL-1ra) expression, function, and cytokinemediated regulation during mycobacterial and schistosomal antigenelicited granuloma formation. J Immunol 1996;156:25032509.
50. Lyadova IV, Tsiganov EN, Kapina MA, Shepelkova GS, Sosunov VV,
Radaeva TV, Majorov KB, Shmitova NS, van den Ham HJ, Ganusov
VV, et al. In mice, tuberculosis progression is associated with
intensive inammatory response and the accumulation of Gr-1 cells
in the lungs. PLoS One 2010;5:e10469.
499