Potential of Host Markers Produced by Infection Phase-Dependent Antigen-Stimulated Cells for the Diagnosis of Tuberculosis in a Highly Endemic Area

Abstract

Background

Recent interferon gamma (IFN-γ)-based studies have identified novel Mycobacterium tuberculosis (M.tb) infection phase-dependent antigens as diagnostic candidates. In this study, the levels of 11 host markers other than IFN-γ, were evaluated in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens, for the diagnosis of TB disease.
Methodology and Principal Findings

Five M.tb infection phase-dependent antigens, comprising of three DosR-regulon-encoded proteins (Rv2032, Rv0081, Rv1737c), and two resucitation promoting factors (Rv0867c and Rv2389c), were evaluated in a case-control study with 15 pulmonary TB patients and 15 household contacts that were recruited from a high TB incidence setting in Cape Town, South Africa. After a 7-day whole blood culture, supernatants were harvested and the levels of the host markers evaluated using the Luminex platform. Multiple antigen-specific host markers were identified with promising diagnostic potential. Rv0081-specific levels of IL-12(p40), IP-10, IL-10 and TNF-α were the most promising diagnostic candidates, each ascertaining TB disease with an accuracy of 100%, 95% confidence interval for the area under the receiver operating characteristics plots, (1.0 to 1.0).
Conclusions

Multiple cytokines other than IFN-γ in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens show promise as diagnostic markers for active TB. These preliminary findings should be verified in well-designed diagnostic studies employing short-term culture assays.

Full text

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Potential of Host Markers Produced by Infection Phase-
Dependent Antigen-Stimulated Cells for the Diagnosis of
Tuberculosis in a Highly Endemic Area
Novel N. Chegou1*, Paulin N. Essone1, Andre G. Loxton1, Kim Stanley1, Gillian F. Black1, Gian D. van der
Spuy1, Paul D. van Helden1, Kees L. Franken2, Shreemanta K. Parida3, Michel R. Klein2,
Stefan H. E. Kaufmann3, Tom H. M. Ottenhoff2, Gerhard Walzl1
1DST/NRF Centre of Excellence for Biomedical Tuberculosis Research and MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human
Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, Stellenbosch University, Tygerberg, South Africa, 2Department of Infectious Diseases, Leiden
University Medical Centre, Leiden, The Netherlands, 3Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany
Abstract
Background: Recent interferon gamma (IFN-c)-based studies have identified novel Mycobacterium tuberculosis (M.tb)
infection phase-dependent antigens as diagnostic candidates. In this study, the levels of 11 host markers other than IFN-c,
were evaluated in whole blood culture supernatants after stimulation with M.tb infection phase-dependent antigens, for the
diagnosis of TB disease.
Methodology and Principal Findings: Five M.tb infection phase-dependent antigens, comprising of three DosR-regulon-
encoded proteins (Rv2032, Rv0081, Rv1737c), and two resucitation promoting factors (Rv0867c and Rv2389c), were
evaluated in a case-control study with 15 pulmonary TB patients and 15 household contacts that were recruited from a high
TB incidence setting in Cape Town, South Africa. After a 7-day whole blood culture, supernatants were harvested and the
levels of the host markers evaluated using the Luminex platform. Multiple antigen-specific host markers were identified with
promising diagnostic potential. Rv0081-specific levels of IL-12(p40), IP-10, IL-10 and TNF-a were the most promising
diagnostic candidates, each ascertaining TB disease with an accuracy of 100%, 95% confidence interval for the area under
the receiver operating characteristics plots, (1.0 to 1.0).
Conclusions: Multiple cytokines other than IFN-c in whole blood culture supernatants after stimulation with M.tb infection
phase-dependent antigens show promise as diagnostic markers for active TB. These preliminary findings should be verified
in well-designed diagnostic studies employing short-term culture assays.
Citation: Chegou NN, Essone PN, Loxton AG, Stanley K, Black GF, et al. (2012) Potential of Host Markers Produced by Infection Phase-Dependent Antigen-
Stimulated Cells for the Diagnosis of Tuberculosis in a Highly Endemic Area. PLoS ONE 7(6): e38501. doi:10.1371/journal.pone.0038501
Editor: Madhukar Pai, McGill University, Canada
Received February 29, 2012; Accepted May 10, 2012; Published June 5, 2012
Copyright: ? 2012 Chegou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Bill & Melinda Gates Foundation through Grand Challenge 6 (BMGFGC6, grant no 37772). Dr. Chegou received
financial support from the Claude Leon Foundation and the South African Medical Research Council during the writing up of this manuscript. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: novel@sun.ac.za
Introduction
The diagnosis of tuberculosis (TB) remains a challenge in
resource-constrained settings. In the absence of culture facilities,
laboratory diagnosis of the disease is often only possible by Ziehl
Neelsen-stained sputum smears, a test whose limitations are well
known [1]. The introduction of the recently developed automated
real-time sputumprocessing
XpertMTB/RIF assay (Cepheid Inc., CA, USA) into clinical
practice, is a significant development as the test yields results
within 2 hours, coupled with the detection of rifampicine
resistance [1]. The relatively high operating costs of the test and
other limitations [2], are factors that hamper its use in resource-
limited settings. Furthermore, the use of sputum-based tests is
problematic in, for example, children and extrapulmonary TB
cases, where appropriate quality sputum samples are difficult to
molecularbeaconassay, the
obtain. Immunodiagnostic techniques could be valuable in such
cases [3,4], especially if they can be developed into rapid, point-of-
care tests.
The most widely used TB immunodiagnostic tests, the
interferon gamma (IFN-c) release assays (IGRAs), have proven
to be useful in the diagnosis of Mycobacterium tuberculosis (M.tb)
infection especially in comparison to the tuberculin skin test (TST)
[5–7]. These tests however, do not discriminate between latent
M.tb infection (LTBI) and active TB disease and are therefore of
limited value in high-burden settings with a high proportion of
LTBI [8]. It has been shown that the detection of host markers
other than IFN-c in M.tb-specific antigen-stimulated whole blood
cell cultures might be a useful approach for discriminating
between LTBI and active TB disease [9–13]. Recently, we
investigated the potential of 118 different M.tb infection phase-
dependent antigens using a diluted whole blood assay, and
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identified antigen candidates – mostly resuscitation promoting
factors (rpfs) and DosR regulon-encoded antigens with potential in
the diagnosis of TB disease, as determined by IFN-c measurement
[14]. Using the Luminex platform, we here evaluated the levels of
12 host markers in culture supernatants that were stimulated with
five of these promising diagnostic antigen candidates (Rv2389c,
Rv0867c, Rv2032, Rv1737c, Rv0081), with the aim of identifying
potentially useful diagnostic markers. We show differential
cytokine production in response to M.tb infection phase-dependent
antigens in participants with and without active TB which warrant
further investigation of their diagnostic potential.
Materials and Methods
Ethics Statement
Ethical approval for this study was obtained from the
Committee for Human Research of the University of Stellenbosch.
All the study participants gave written informed consent for
participation in the study.
Study Participants
Participants enrolled into this study were recruited as part of
the on-going Bill & Melinda Gates Foundation-funded Grand
Challenges in Global Health (BMGF GC6-74) study (http://
www.biomarkers-for-tb.net/) and have previously been described
in [9,14]. Briefly, all participants were recruited from the
Ravensmead/Uitsig community, a high TB-endemic community
[15] in Cape Town, South Africa, between October 2006 and
April 2007. All TB patients were self-reporting, untreated cases
with a first episode of TB and were acid fast bacilli (AFB)-
positive on two sputum smears. Household contacts (HHCs) had
been living in the same house as an adult TB case who was
diagnosed not more than 2 months before recruitment of the
contact. All HHCs had normal chest X-rays and AFB negative
assisted sputum samples. All participants were between 18 and
60 years old, had negative HIV results (Abbot DetermineTM
HIV 1/2; Abbott, Wiesbaden, Germany), and gave written
informed consent for participation in the study. Exclusion
criteria for all participants included HIV infection, previous or
current TB treatment, serious concomitant chronic conditions,
steroid therapy within the past 6months and pregnancy. After
collection of demographic data and completion of a clinical
questionnaire, 10 ml of heparinized blood was collected from all
participants and transported within 2 hours of collection to the
laboratory where a 7-day whole blood assay (WBA) was
performed as described in [14]. The TST, using 2 TU PPD
RT23 (Statens Serum Institute, Copenhagen, Denmark), was
performed on all HHCs (Mantoux method) after blood draw.
Antigen Screening and Selection of Supernatants for
Multiplex Cytokine Evaluation
A total of 118 M.tb infection phase-dependent antigens were
evaluated as possible diagnostic candidates, based on the detection
of IFN-c responses in 7-day whole blood culture supernatants as
reported previously [14]. Many M.tb infection phase-dependent
antigens with diagnostic potential were identified. However, none
of these antigens sufficed for diagnosis of TB disease with an
accuracy of 100%, and such high accuracy was also not obtained
when antigens were used in combinations [14]. We therefore
investigated the levels of 11 alternative host markers in addition to
IFN-c, in aliquots of supernatants from whole blood cultures
stimulated with the two most promising rpfs (Rv0867c and
Rv2389c) and three DosR regulon-encoded antigens (Rv2032,
Rv0081 and Rv1737c), alongside an unstimulated control and
ESAT-6/CFP-10-stimulated cultures, using a customized Lumi-
nex multiplex assay.
All of the antigens evaluated in this study were recombinant
proteins. As described previously [16], endotoxin contents in all
antigens were below 50 IU/mg recombinant protein as tested by
Limulus Amebocyte Lysate assay (Cambrex, East Rutherford, NJ),
and all the proteins were further tested to exclude non-specific T
cell stimulation and cellular toxicity in IFN-c release assays.
Luminex Multiplex Immunoassay
The levels of 12 host markers [epidermal growth factor, (EGF);
fractalkine; interferon (IFN)-a2; IFN-c; interleukin (IL) -4; IL-10;
IL-12(p40); transforming growth factor (TGF)-a; tumour necrosis
factor (TNF)-a; vascular endothelial growth factor (VEGF);
interferon-inducible protein (IP)-10; RANTES] were determined
in the selected supernatants using customized Milliplex kits (Merck
Millipore, St. Charles, Missouri, USA), on the Bio Plex platform
(Bio PlexTM, Bio Rad Laboratories). This was done according to
manufacturer’s instructions (Merck Millipore). Following previous
optimizations, all supernatants were tested undiluted, in a blinded
manner. All analyte levels in the quality control reagents of the kits
were within the expected ranges. The standard curve for all
analytes ranged from 3.2–10000 pg/ml. Bio-Plex Manager
Software, version 4.1.1 was used for the analysis of bead median
fluorescence intensity.
Statistical Analysis
Statistical differences in analyte levels were evaluated by the
Mann Whitney U test for non-parametric data analysis. Cut-off
levels for differentiating between TB disease and no-TB were
ascertained by receiver operating characteristics (ROC) analysis
based on the highest likelihood ratio. The predictive abilities of
combinations of antigen–specific host markers for TB disease and
no-TB were investigated by performing best subsets general
discriminant analysis (GDA), with leave-one-out cross validation as
described previously [9]. A 5% significance level was used as
guideline for determining significant associations. Data were
analyzed using the GraphPad prism, version 5.00 for Windows
(GraphPad Software, San Diego, California, USA) and Statistica
software (Statsoft, Ohio, USA).
Results
A total of 43 individuals (15 pulmonary TB cases and 28 HHCs)
were included in the study. All antigens were evaluated in all of the
15 TB cases and in different HHCs (total of 15 for each antigen)
from the 28 contacts that were randomly selected from the sample
bank of the parent study. The mean age (6standard deviation) of
all study participants was 31.5615.9 years and 60.5% (26/43)
were males. Of the 28 HHCs, 3 (10.7%) were not available for
TST reading. Of the 26 with available TST results, 88.5% (23/26)
were positive (10-mm cut-off), with a 2068 mm mean (6standard
deviation) induration of all 26 participants.
Potential of Host Markers Produced by Unstimulated and
ESAT-6/CFP-10-stimulated Blood Cells in Discriminating
Individuals with or without TB Disease
When analyte levels detected in the unstimulated control
supernatants in TB patients were compared to levels obtained in
HHCs, significant differences were obtained for EGF, IFN-a2 and
IL-4. After ROC analysis, however, the area under the ROC
curve (AUC) was $0.70 only for EGF and IL-4. The sensitivities
of the two analytes (EGF and IL-4) for TB disease were both
,50%, but with specificities $96% (Table S1). The unstimulated
Antigen-Specific Host Markers for TB
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control levels for the different host markers were subtracted from
the antigen-stimulated responses for the respective study partici-
pants prior to further analysis of the data.
When ESAT-6/CFP-10 stimulated analyte levels were com-
pared between the TB cases and HHCs, significant differences
were obtained for EGF, TGF-a and TNF-a, consistent with
previous observations for EGF and TGF-a in overnight whole
blood assays [9]. After ROC analysis, the AUC for all of these
three markers were $0.70. The sensitivities of the three analytes
for TB disease were all ,50%, but with specificities .96%
(Figure 1, Table S1).
Potential of Markers Produced by Rv0867c- and Rv2389c-
stimulated Blood Cells in Discriminating Individuals with
or without TB Disease
Two of the five M.tb rpfs (Rv0867c and Rv2389c) were
evaluated in this study. Although IFN-c responses to the two
rpfs were significantly higher in HHCs in comparison to TB
cases as previously observed in the IFN-c ELISA based study
[14], the median levels of most of the other markers were
higher in TB cases (Table S1). Of the other 11 host markers
evaluated in Rv0867c-stimulated supernatants, TGF-a was the
only additional marker (other than IFN-c) that was significantly
different between the TB cases and HHCs (Table S1). For
Rv2389c-stimulated supernatants, significant differences between
the TB cases and HHCs were obtained for five additional
markers, namely, TGF-a, TNF-a, VEGF, IL-10 and RANTES.
Unlike IFN-c, the median levels of these markers were higher in
TB cases (Table S1). After ROC analysis, the AUC for these
markers (discriminating between TB disease and no-TB) was
$0.70, with TGF-a differentiating between the two groups with
both sensitivity and specificity .92% (Table S1, Figure 2).
Potential of Host Markers Produced by Rv2032-, Rv1737c-
and Rv0081-stimulated Blood Cells in Discriminating
Individuals with or without TB Disease
Three DosR-regulon-encoded antigens (Rv2032, Rv1737c and
Rv0081) were evaluated in this study. The levels of eight out of the
12 markers evaluated in this study (IFN-c, IFN-a2, IL-12(p40), IP-
10, TNF-a, VEGF, IL-10 and RANTES), were significantly
higher in the HHCs than the TB cases in Rv0081-stimulated
supernatants (Table S1). AUC was $0.84 for all of these analytes,
with IP-10, IL-10, TNF-a and IL-12(p40) discriminating between
TB cases and HHCs with a sensitivity and specificity of 100%
(Table S1, Figure 3).
For Rv2032, the levels of seven markers (fractalkine, IL-12(p40),
TGF-a, TNF-a, VEGF, IL-10, RANTES) were significantly
higher in TB cases than HHCs. These seven analytes discrimi-
nated between TB and no-TB disease with an AUC $0.75, and
TNF-a, TGF-a and IL-10 ascertaining TB disease with both
sensitivity and specificity of $85% (Figure 4, Table S1).
Similarly, Rv1737c-specific levels of IL-10, TGF-a, TNF-a, IL-
12(p40) and EGF were significantly higher in the TB cases in
comparison to HHCs. After ROC analysis, the AUC for all five
analytes was $0.72 (Figure 5, Table S1).
Figure 1. Host markers detected in ESAT-6/CFP-10-stimulated whole blood cell cultures in TB and no-TB cases and receiver
operating characteristics (ROC) plots showing the accuracies of these markers in discriminating between TB disease and no-TB.
Only analytes that discriminated between TB and no-TB disease with AUC $0.70 are shown. Error bars in the scatter dot plots represent the median
analyte levels. AUC = Area under the ROC curve.
doi:10.1371/journal.pone.0038501.g001
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Figure 2. Host markers detected in Rv2389c stimulated whole blood cell cultures of TB and no-TB cases and receiver operating
characteristics (ROC) plots showing the accuracies of these markers in discriminating between TB disease and no-TB. Representative
plots for analytes that discriminated between TB and no-TB disease with AUC $0.70, are shown. Error bars in the scatter plots represent the median
analyte levels. AUC = Area under the ROC curve.
doi:10.1371/journal.pone.0038501.g002
Figure 3. Host markers detected in Rv0081-stimulated whole blood cell cultures of TB and no-TB cases and receiver operating
characteristics (ROC) plots showing the accuracies of these markers in discriminating between TB disease and no-TB. Representative
plots for the most potentially useful analytes are shown. The unstimulated control levels of the markers with negative responses were higher than the
analyte levels detected in Rv0081 stimulated culture supernatants. Error bars in the scatter plots represent the median analyte levels. AUC = Area
under the ROC curve.
doi:10.1371/journal.pone.0038501.g003
Antigen-Specific Host Markers for TB
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Figure 4. Host markers detected in Rv2032-stimulated whole blood cell cultures of TB and no-TB cases and receiver operating
characteristics (ROC) plots showing the accuracies of these markers in discriminating between TB disease and no-TB. Representative
plots for analytes that discriminated between TB and no-TB disease with AUC $0.70 are shown. The unstimulated control levels of the markers with
negative responses were higher than the analyte levels detected in Rv2032 stimulated culture supernatants. Error bars in the scatter plots represent
the median analyte levels. AUC = Area under the ROC curve.
doi:10.1371/journal.pone.0038501.g004
Figure 5. Host markers detected in Rv1737c-stimulated whole blood cell cultures and receiver operating characteristics (ROC) plots
showing the accuracies of these markers in discriminating between TB disease and no-TB. Only the top 4 Rv1737c-specific analytes are
shown. Error bars in the scatter dot plots represent the median analyte levels. AUC = Area under the ROC curve.
doi:10.1371/journal.pone.0038501.g005
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Utility of Analyte Combinations in Discriminating
between TB and no-TB Disease
Evidence has been presented that different antigens are
differentially recognised in different populations [17]. Considering
that even the most promising antigens identified in that study were
not recognized in all populations that were studied, and that useful
diagnostic tests should perform reliably in all populations, we
evaluated the performance of antigens in combination. A general
discriminant analysis (GDA) [9] procedure was performed in two
steps, firstly for the unstimulated and ESAT-6/CFP-10-stimulated
responses, and then for all other antigen (Rv0867c, Rv2389c,
Rv0081, Rv2032 and Rv1737c)-induced markers.
For combinations between the unstimulated control and
ESAT-6/CFP-10-specificanalytes,
marker combination models resulted in the accurate prediction
of $80% of the TB cases and $90% of the HHCs after leave-
one-out cross validation (data not shown). The top antigen–host
marker combination (unstimulated EGF + unstimulated fractalk-
ine + unstimulated IFN-a2+ unstimulated IL-4+ ESAT-6/CFP-
10-specific RANTES) accurately predicted 87% of TB cases and
100% of HHCs. The host markers that were included most
frequently among the top 20 GDA -marker combinations were
unstimulated EGF and unstimulated IFN-a2 (Figure 6). For the
combinations between Rv0867c, Rv2389c, Rv0081, Rv2032 and
Rv1737c-specific analyte levels, all the top 20 five-antigen-
specific host marker combinations accurately predicted all (100%)
of the TB cases and HHCs after leave-one-out cross validation
(data not shown). The most predictive analytes in the top 20
analytecombinationmodels
12(p40), Rv1737c-specific EGF and Rv0081-specific TGF-a
levels (Figure 6).
differentfive-antigen-host
includedRv0081-specific IL-
Discussion
IFN-c remains the primary marker in T-cell stimulation assays,
due to its critical role in the development of M.tb-specific acquired
immune responses [18]. In this study, we show that stimulation of
whole blood cells from TB patients and HHCs with different M.tb
infection phase-dependent antigens, results in the production of
multiple host markers other than IFN-c. In addition to confirming
results obtained with IFN-c in previous studies, we show that
several, non-canonical analytes are promising markers of active
TB disease. M.tb DosR regulon-encoded Rv0081 antigen-specific
induced levels of IL-12(p40), IP-10, IL-10 and TNF-a were the
most promising diagnostic candidates in this study. Combining
different M.tb antigens and host markers may improve diagnostic
ability substantially.
Much progress has been made in the development of new and
more effective diagnostic tools for TB, notably IGRAs and the
more recently developed XpertMTB/RIF assay. In spite of this
progress, the diagnosis of TB disease in resource-constrained
settings, which often bear the highest TB and HIV co-infection
burden, especially Sub-Saharan Africa [19,20], remains challeng-
ing. This is mainly because of the continued reliance in these
settings on smear microscopy and empirical clinical diagnosis due
to the lack of proper laboratory diagnostic tools [19]. Such settings
would benefit from a rapid and robust field-friendly point-of-care
test, especially if such a test proves to be highly accurate in the
diagnosis of active TB disease in areas with concurrently high
LTBI rates. The host markers evaluated in this study are pro-
inflammatory and anti-inflammatory markers that have been
widely investigated for different purposes in different infectious
diseases, including TB. The uniqueness of our study, however, is
the fact that these markers were evaluated in supernatants of
whole blood cultures that were stimulated with recently identified
M.tb infection phase-dependent antigens, and that these markers
are of potential value for the diagnosis of TB disease in a highly
endemic setting. This study was conducted in an area where the
prevalence of LTBI is very high, as evidenced by high IGRA
positivity in previous studies [6,7,9,17], and the .88% TST
positivity of the non TB cases in this study. Thus, these novel M.tb
antigen-specific markers may be useful in discriminating between
LTBI and active TB disease.
The concept that stimulation of whole blood cells from M.tb-
infected or -diseased individuals with M.tb antigens results in the
secretion of multiple host markers, that could be useful diagnostic
markers is not new as such markers have been investigated in other
studies [9,12,13,21–29], including studies on bovine TB [30].
These mostly ESAT-6/CFP-106 TB7.7 -based studies have
implicated some of the markers investigated in this study, notably
TNF-a and IP-10 (reviewed in [31]) [22,25–27,29] as adjunct
markers for use singly or in combination with IFN-c in the
diagnosis of M.tb infection; or as potential candidates for
discriminating between LTBI and active TB disease (IP-10,
TNF-a, IL-12(p40), EGF, TGF-a, and VEGF) [9–13]. The results
obtained for the ESAT-6/CFP-10-specific levels of EGF, TGF-a,
and the unstimulated EGF responses in this study are in agreement
with previous observations in the same study population [9].
Stimulation of whole blood with other antigens such as TB10.4,
coupled with the detection of host markers such as TNF-a, IL-17,
and IL-12(p40) has also been shown to be potentially useful in the
diagnosis of TB disease in a West African setting [13]. Out of 29
host markers, including most of the markers evaluated in this
study, that were investigated for discrimination between LTBI and
TB disease in children in a low burden setting however, none of
the markers, with the exception of IL-2, was significantly different
between latently infected children and those with TB disease [11].
It is not known whether this difference in the studies could only be
explained by factors such as age of study participants (children
versus adults), geographical location, bacterial strain differences
and other factors including genetic differences in HLA genes [32].
It is worth mentioning however, that data on host markers for
discriminating between TB disease and LTBI are currently very
limited, extremely heterogeneous, and such studies have so far
been performed only in limited participant numbers, partly
because of the cost of multiplex immunoassay kits as acknowl-
edged in [9,22]. Furthermore, the potential for false discoveries
also exists, given the large numbers of markers that are often
evaluated and the small patient numbers in these studies. More
data on the most promising markers that have been described to
date are needed before their diagnostic potential can be verified.
However, suitable selection criteria for future qualification of
promising markers are currently not defined but will be absolutely
necessary to allow affordable evaluation of candidate markers.
Little is known about the relatively new M.tb infection phase-
dependent antigens investigated in this study (Rv0081, Rv2032,
Rv0867c, Rv2389c, Rv1737c), notably about their immunogenic-
ity in LTBI or TB disease. In the few studies that have investigated
these antigens however, it has been shown that IFN-c responses to
both the rpfs and DosR regulon-encoded antigens are strongly
associated with latency [33–35]. Furthermore, the rpfs and DosR
regulon-encoded antigens have also been shown to elicit marked
CD4 and CD8 T cell responses in vitro, in long-term latently
infected individuals [35,36]. Our recent report [14] based on IFN-
c measurement in culture supernatants by ELISA implicated
Rv0081, Rv2032, Rv0867c, and Rv2389c as promising diagnostic
antigen candidates. In spite of the promise shown by these antigens
as possible TB diagnostic candidates none of the antigens
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discriminates between TB and absence of TB disease with both
sensitivity and specificity .85% in IFN-c based tests and this
degree of accuracy is not achieved even when antigens are
evaluated in combinations of four [14]. We have, however, shown
for the first time that unlike IFN-c for which higher levels are
observed in latently infected individuals upon stimulation of whole
blood with these antigens, the levels of most other markers were
higher in patients with active TB disease with the exception of
Rv0081-specific markers, which were generally higher in the
HHCs. Work done with other DosR regulon-encoded antigens,
such as Rv2628, although not investigated in this study, has shown
that responses against this antigen are associated with cured TB
and remote latent infection [37], and higher frequencies of T cell
responses to this antigen occur at the site of disease [38]. Our data
indicate that measurement of host markers such as IL-12(p40), IP-
10, IL-10 and TNF-a upon stimulation with even a single antigen,
such as Rv0081, could be sufficient in diagnosing TB disease with
high accuracy in a setting with high LTBI.
Of all the 12 markers evaluated in this study, the levels of 11
(IL-12(p40), IFN-c, IP-10, TNF-a, IL-10, TGF-a, IFN-a2,
RANTES, VEGF, fractalkine, EGF) after background correction,
were significantly different between the TB and no TB cases
Figure 6. Number of times each analyte was included in the best 20 general discriminant analysis models that best classified the TB
cases and HHCs into their respective groups. A, Analytes detected in the unstimulated and ESAT-6/CFP-10 stimulated supernatants. B, Analytes
detected in Rv0867c-, Rv2389c-, Rv2032-, Rv0081- and Rv1737c-stimulated supernatants.
doi:10.1371/journal.pone.0038501.g006
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following stimulation with at least one of the six antigens
investigated. IL-12(p40), TNF-a, IL-10 and IP-10, a chemokine
produced by many cell types including monocytes, endothelial
cells, neutrophils amongst others in response to IFN-c [31], were
amongst the best candidate diagnostic markers identified in this
study. The median levels of these markers in Rv0081 stimulated
supernatants, just like the levels of IFN-c, were higher in the non
TB cases. From what is known about the biological roles of these
markers (all Th1 immune response and/or granuloma formation
favourable signals; with the exception of IL-10 which is anti-
inflammatory) [39,40], it is not surprising that the induced levels
of the markers in Rv0081 stimulated supernatants all correlated
significantly with IFN-c (r, 0.40–0.62; p, 0.02–,0.001). Although
the levels of these markers were higher in the non TB cases in
Rv0081 stimulated supernatants, higher levels of the markers
were observed in the TB cases upon stimulation with the other
DosR regulon-encoded antigens (Rv2032 and Rv1733c), and
correlations between the four markers (IL-12p40, IL-10, IP-10,
TNF-a) and IFN-c were only significant for IL-12(p40) (r, 0.43;
p, 0.02) in Rv2032 stimulated supernatants, and IP-10 (r, 0.5; p,
,0.01) in Rv1737c stimulated supernatants. The generally
higher Rv2032- and Rv1733c-specific marker responses in TB
cases were more similar to what was observed in the rpf
(Rv2389c and Rv0867c) -stimulated supernatants. The meaning
of this differential marker induction pattern, even in antigens that
are expressed in the same phase of M.tb infection, is not clear as
data on these new antigens is still scanty. IFN-c responses against
all the antigens however, were as observed in the recent IFN-c
ELISA based study on the same participant cohort [14], and in
agreement with previous observations [33–35]. Future validation
studies will determine which of the antigen–specific markers
analyzed here possesses the best diagnostic potential, given that
the diagnostic potential of these M.tb antigens and host markers
can be maintained in overnight assays. These antigen–specific
host markers may provide the basis for the development of a
novel TB diagnostic test, highly suitable for high TB burden
areas.
The molecular beacon assays such as the GeneXpert test
provide results within 2 hours. Yet, the strength of an immuno-
diagnostic modality is that such an assay could be more practical
in high TB burden and resource-limited settings, considering the
obstacles involved in the use of the GeneXpert and similar tests
that rely on advanced laboratory equipment as highlighted by
Trebucq et al [2]. If the diagnostic potential of the antigen specific
host markers investigated in this preliminary study is maintained in
larger validation studies, T cell based immunodiagnostic modal-
ities might be developed, that involve the coating of blood
collection tubes with the antigens for short-term culture, as is
currently done with the Quantiferon TB Gold In Tube system.
Antibodies against the target host markers could then be
incorporated into a dip-stick-like test membrane which would
then be inserted into the supernatant after culture and results read
in 10 to 20 minutes. Such lateral flow test devices are currently
being evaluated for the diagnosis of many infectious diseases
including TB [41–44], and these assays have the potential to detect
multiple host markers, including some of the markers evaluated in
this study, in culture supernatants [43]. Although such a diagnostic
modality will only yield results within 24 hours after the patient’s
visit, it might prove cheaper and more suitable in remote settings
than the GeneXpert and conventional M.tb culture which has
been the gold standard for a long time, but yet unavailable in these
resource-limited settings. Such a test would also be beneficial to
individuals that have difficulty in providing satisfactory sputum
samples for microbiological and other tests such as children and
individuals with extrapulmonary TB.
One concern relating to the development of lateral flow based
tests is the ability to detect markers that are produced in low
amounts. It has been shown that cytokine responses can be
enhanced by addition of cytokines such as IL-7 [45] and IL-12
[46] into cultures and through the delivery of the antigens using
vectors such as the Bordetella pertusis adenylate cyclase vector [47],
and other adjuvants [48]. Lateral flow tests using upconverting
phospor technology were shown to detect IFN-c in culture
supernatants with sensitivity below 2 pg/ml and were more
sensitive than ELISA in detecting Shistosoma circulating antigens
[44]. Such considerations will only become relevant if diagnostic
ability of such markers is validated in appropriate larger studies.
As previously reported [14], a serious concern in the possible
use of new antigens such as those investigated in this study for
diagnostic purposes is a lack of specificity for M.tb. Some DosR
regulon-encoded antigens have been shown to be expressed in
BCG [49], which could potentially affect the value for diagnosis in
BCG-vaccinated populations. However, BCG vaccination was
found not to induce T cell responses to DosR regulon-encoded
antigens, likely because BCG fails to establish long-lasting latent
infection and therefore may not express these antigens in
vaccinated individuals [50]. Future studies also need to evaluate
specificity in lung inflammation other than TB as levels of several
cytokines in unstimulated and/or ESAT-6 stimulated whole blood
samples have been shown to not be useful in discriminating
between TB disease and pneumonia [23].
Study Limitations
The main limitations of our study include the small sample size
and the use of a 7-day whole blood assay instead of an overnight
assay which would be more suitable for a diagnostic test. Another
limitation of our study is the potential of reporting a significant
finding which occurred by chance, given that 12 host markers
were evaluated in 5 different M.tb infection phase-dependent
antigen-stimulated supernatants. Such a risk applies in all
biomarker discovery studies regardless of the discovery platform
used. One corrective measure that is usually employed during
statistical analysis is correcting for multiple comparisons. The
main analytical procedure employed in this study was ROC
analysis, a method in which no hypothesis testing is done, the
decisions rather resulting from likelihood ratios [51]. Not
correcting for multiple comparisons in this study however, may
be a concern in GDA as the best subsets method (applied in this
study) does generate different analyte combinations. However, as
we did not compare different combinations of analytes and rather
evaluated the frequency of inclusion of individual markers into the
top 20 models that were generated by GDA (Figure 6), correction
for multiple comparisons does not apply.
Future evaluation of the antigens and host markers identified in
this study should be done in larger numbers of participants, in
overnight assays to detect effector cell responses, and preferably
using a prospective study design. Future studies should also
evaluate the antigens and host markers in children, immunocom-
promised individuals, extrapulmonary TB cases, and also in
individuals with other lung diseases [23]. A diagnostic modality
derived from these antigens and host markers would be highly
valuable for TB control if the host markers could be incorporated
into a rapid, lateral flow test device.
Conclusions
In conclusion, the measurement of host markers (other than
IFN-c) in supernatants after stimulation with M.tb infection phase-
Antigen-Specific Host Markers for TB
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Page 9

dependent antigens promises to be a useful method of diagnosing
TB disease. Rv0081-specific levels of IP-10, IL-12(p40), IL-10 and
TNF-a, Rv2389c-specific levels of TGF-a, and Rv2032-specific
levels of TNF-a showed the most promise as TB diagnostic
candidates. The results of this study are preliminary and warrant
further investigation in larger studies and in other settings.
Supporting Information
Table S1
ranges (in parentheses) elicited upon stimulation of whole blood
cells with different M. tuberculosis infection phase-dependent
antigens and abilities to discriminate between pulmonary TB
disease (in 15 cases) and no TB (in 15 household contacts). AUC,
area under the curve; 95% CI=95% confidence interval. The p-
values shown are Mann Whitney U test P values for differences
between TB cases and HHCs. The cut-off values are for the
Median levels of all analytes evaluated (pg/ml) and
sensitivity and specificity for TB disease. Antigens and host
markers were sorted according to AUC (highest to lowest) for
discriminating between TB and no-TB disease.
(XLS)
Acknowledgments
We are grateful to Professor Martin Kidd of the Centre for Statistical
Consultation of the University of Stellenbosch for statistical advice and
analysis of the data.
Author Contributions
Conceived and designed the experiments: NNC GFB SKP MRK SHK
THO GW. Performed the experiments: NNC PNE AGL KS KLF.
Analyzed the data: NNC KLF GFB GvdS GW. Contributed reagents/
materials/analysis tools: GFB GvdS PvH THO SHK GW. Wrote the
paper: NNC PVH MRK SHK THO GW.
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