Tuberculosis antigen‐specific immune responses can be detected using enzyme‐linked immunospot technology in human immunodeficiency virus (HIV)‐1 patients with advanced disease

Article (PDF Available)inClinical & Experimental Immunology 150(2):238-44 · November 2007with22 Reads
DOI: 10.1111/j.1365-2249.2007.03477.x · Source: PubMed
Abstract
There are limited data on the efficacy of T cell-based assays to detect tuberculosis (TB) antigen-specific responses in immune-deficient human immunodeficiency virus (HIV) patients. The aim of this study is to determine whether TB antigen-specific immune responses can be detected in patients with HIV-1 infection, especially in those with advanced disease (CD4 T cell count < 300 cells/microl). An enzyme-linked immunospot (ELISPOT) assay, which detects interferon (IFN)-gamma secreted by T cells exposed to TB antigens, was used to assess specific immune responses in a prospective study of 201 HIV-1-infected patients with risk factors for TB infection, attending a single HIV unit. The performance of the ELISPOT assay to detect TB antigen-specific immune responses is independent of CD4 T cell counts in HIV-1 patients. The sensitivity and specificity of this assay for the diagnosis of active tuberculosis does not differ significantly from values obtained in immunocompetent subjects. The negative predictive value of the TB ELISPOT test is 98.2%. A positive predictive value of 86% for the diagnosis of active tuberculosis was found when the combined number of early secretory antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) IFN-gamma spots to CD4 T cell count ratio was > 1.5. TB antigen-specific immune responses can be detected in HIV patients with low CD4 T cell counts using ELISPOT technology in a routine diagnostic laboratory and is a useful test to exclude TB infection in immune-deficient HIV-1 patients. A combination of TB antigen-specific IFN-gamma responses and CD4 T cell counts has the potential to distinguish active tuberculosis from latent infection.
Tuberculosis antigen-specific immune responses can be detected
using enzyme-linked immunospot technology in human
immunodeficiency virus (HIV)-1 patients with advanced disease
S. A. Clark,* S. L. Martin,
A. Pozniak,
A. Steel,* B. Ward,
J. Dunning,
D. C. Henderson,
M. Nelson,
B. Gazzard
and
P. K e l l e h e r
*
†‡
*Department of Immunology, Imperial College,
Chelsea & Westminster Hospital, London, UK,
Division of Immunology Hammersmith
Hospitals NHS Trust, London, UK, and
HIV/GUM Directorate, Chelsea & Westminster
NHS Foundation Trust, London, UK
Summary
There are limited data on the efficacy of T cell-based assays to detect tuber-
culosis (TB) antigen-specific responses in immune-deficient human immuno-
deficiency v i rus (HIV) patients. The aim of this study is to determine whether
TB antigen-specific immune responses can be detected in patients with HIV-1
infection, especially in those with advanced disease (CD4 T cell count < 300
cells/ml). An enzyme-linked immunospot (ELISPOT) assay, which detects
interferon (IFN)-g secreted by T cells exp osed to TB antigens, was used to
assess specific immune responses in a prospective study of 201 HIV-1-infected
patients with risk factors for TB infection, attending a single HIV unit. The
performance of the ELISPOT assay to detect TB antigen-specific immune
responses is independent of CD4 T cell counts in HIV-1 patients. The sensi-
tivity and specificity of this assay for the diagnosis of active tuberculosis does
not differ significantly from values obtained in immunocompetent subjects.
The negative predictive value of the TB ELISPOT test is 98·2%. A positive
predictive value of 86% for the diagnosis of active tuberculosis was found
when the combined number of early secretory antigen target-6 (ESAT-6) and
culture filtrate protein-10 (CFP-10) IFN-g spots to CD4 T cell count ratio
was > 1·5. TB antigen-specific immune responses can be detected in HIV
patients with low CD4 T cell counts using ELISPOT technology in a routine
diagnostic laboratory and is a useful test to exclude TB infection in immune-
deficient HIV-1 patients. A combination of TB antigen-specific IFN-g
responses and CD4 T cell counts has the potential to distinguish active
tuberculosis from latent infection.
Keywords: culture filtrate protein-10 (CFP-10)
, early secretory antigen
target-6 (ESAT-6), HIV-1 infection, interferon gamma (IFN-g), tuberculosis
Accepted for publication 2 July 2007
Correspondence: Dr P Kelleher, Department
of Immunology, Imperial College, Chelsea &
Westminster Hospital, London, UK.
E-mail: p.kelleher@imperial.ac.uk
Introduction
Tuberculosis is the most common opportunistic infection
and the leading cause of death worldwide in human immu-
nodeficiency virus (HIV)-1 infected patients [1]. The
clinical and radiological features of tuberculosis (TB) are
influenced by the degree of HIV-1 related immune defi-
ciency, especially in patients with CD4 T cell counts < 300
cells/ml [2]. The performance of diagnostic tests such as
sputum microscopy and tuberculin skin tests (TST)
declines in patients with reduced CD4 T cell counts. There
is a pressing need for rapid, sensitive tests to identify both
active and latent TB infection to control tuberculosis in
HIV-1 infection and other high-risk populations. A number
of studies have shown that T cell assays that detect inter-
feron (IFN)-g secretion in response to TB-specific antigens,
early secretory antigen target-6 (ESAT-6) and culture filtrate
protein-10 (CFP-10), are significantly better than TST for
the diagnosis of active and latent TB infection in immuno-
competent individuals [3–8]. There is limited information
on the performance of IFN-g release assays in patients with
HIV-1 infection. In this study, we investigate the perfor-
mance of an enzyme-linked immunospot (ELISPOT) assay
(T-SPOT.TB; Oxford Immunotec, Oxford, UK) assay to
detect TB antigen-specific immune responsiveness in HIV-1
patients.
Clinical and Experimental Immunology
ORIGINAL ARTICLE doi:10.1111/j.1365-2249.2007.03477.x
238
© 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 238–244
Material and methods
Study population
HIV-1-positive patients attending a single HIV unit in
London were screened for TB infection using the T-SPOT.
TB ELISPOT assay. The inclusion criteria for this study were
individuals with either (1) symptoms, signs and X-ray find-
ings suggestive of active tuberculosis or (2) asymptomatic
infection with the following TB risk factors: residence in a
TB endemic area for more than 3 months, TB contact, intra-
venous drug use, diabetes mellitus, chronic renal failure,
current immunosuppressive drug therapy. Absolute CD3,
CD4 and CD8 T cell counts and percentages were deter-
mined using 100 ml of ethylenediamine tetraacetic acid
(EDTA) blood samples by four-colour flow cytometric
analysis using a Cytomics FC500 flow cytometer (Beckman
Coulter Inc., High Wycombe, UK). Plasma viral load was
measured using branched DNA amplification technology
(Bayer Healthcare, Tarrytown, NY, USA) with a lower limit
of detection of 50 copies/ml.
The first 70 patients recruited received tuberculin skin
tests (Heaf or Mantoux) using the equivalent of 2 U of
tuberculin. However, restrictions on the supply of skin test
reagents and authorized personnel resulted in premature
discontinuation of this arm of the study. A positive Mantoux
test result was defined as skin induration of more than 5 mm
in diameter [9] and a positive Heaf test was a grades 2–4
reaction [10]. Clinical information such as age, sex, ethnic
background and risk factors for TB infection were recorded.
Individuals with clinical features or chest X-ray features sug-
gestive of active tuberculosis had three sputum/bronchial
alveolar lavage (BAL) samples requested for acid fast bacilli
(AFB) stains and TB cultures. Further investigations, such as
TB blood, spinal fluid and tissue cultures, were performed as
indicated clinically. Asymptomatic patients with a positive
TB ELISPOT test were referred to the TB clinic for further
investigations to exclude active TB disease. One individual
(P. K.) reviewed the medical notes of all patients studied for
at least 3 months after the TB ELISPOT assay to ascertain
clinical outcomes. Active tuberculosis was defined as AFB
smear-positive or positive culture for Mycobacterium tuber-
culosis, while probable tuberculosis was defined as clinical,
radiological features consistent with TB and objective symp-
tomatic or radiological improvement on standard 6-month
anti-TB therapy in patients with clinically suspected disease.
Asymptomatic HIV patients with no evidence of active TB
but who had TB risk factors as defined above and who were
not receiving TB chemotherapy were defined as potential
latent TB infection. The study was conducted in accordance
with local ethical approval (Riverside Research Ethics
Committee, REC no. 04/Q0401/108).
The T-SPOT.TB ELISPOT assay was performed according
to the manufacturer’s instructions. Viable peripheral blood
mononuclear cells (PBMC) at a concentration of 2·5 ¥ 10
5
were added to 96-well membrane-bottomed plates precoated
with anti-IFN-g antibodies. Four wells were used for each
patient: a positive control well to which phytohaemaggluti-
nin (PHA) was added, a negative control well which con-
tained medium and two wells which contained ESAT-6 or
CFP-10 peptide pools. Plates were incubated for 16–20 h at
37°C with 5% CO
2
, washed with phosphate-buffered saline
(PBS) and developed using an anti-IFN-g antibody conju-
gate and substrate to detect the presence of secreted IFN-g.
The number of spots was then counted using an ELISPOT
reader. Positive, anergic (less than 80 IFN-g spots/10
6
cells
following PHA stimulation) and indeterminate results were
defined according to the manufacturer’s instructions.
Statistical analysis
Univariate correlations between T cell counts and PHA/TB
antigen-specific induced IFN-g responses were assessed
using Spearman’s rank correlation tests. Differences between
patients with active/presumptive TB and latent TB were
tested for statistical significance using the Mann–Whitney
U-test. Agreement of the results obtained by TB ELISPOT
and TST was determined by calculation of Cohen’s
k-coefficient. All statistical calculations were performed with
spss version 14 (SPSS Inc., Chicago, IL, USA).
Results
We evaluated 201 HIV-1 seropositive subjects for TB infec-
tion over a period of 24 months (Table 1): 154 symptomatic
patients who presented either with a history of acute respi-
ratory illness or radiological features suggestive of active
Table 1 . Demographic and clinical characteristics of human immuno-
deficiency virus-1 infected patients who received a tuberculosis enzyme-
linked immunospot and tuberculin skin tests (TST).
Characteristics
Total group
(n = 201)
TST group
(n = 72)
Age, median (IQR) 40 (33–46) 40 (33–46)
Male, no. (%) 119 (59) 41 (57)
Ethnic origin
African, no. (%) 102 (51) 39 (54)
White, no. (%) 69 (34) 25 (35)
Asian/other, no. (%) 30 (15) 8 (11)
CD4Tcellcount
Median (IQR) 213 (77–367) 261 (120–455)
< 300 cells/ml, no. (%) 129 (64) 39 (54)
< 200 cells/ml, no. (%) 96 (48) 25 (35)
< 100 cells/ml, no. (%) 61 (30) 16 (22)
BCG,† no. (%) n.d. 49 (68)
ART, no. (%) 118 (59) 43 (60)
Viral load
< 50 copies/ml, no. (%) 87 (43) 35 (49)
†Bacille Calmette–Guérin (BCG) vaccination status determined on
the basis of clinical history and the presence of a scar. IQR: interquartile
range; n.d. not determined; ART: anti-retroviral therapy.
Immune responses to TB antigens in HIV
239
© 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 238–244
tuberculosis and 47 asymptomatic patients who were
screened for latent TB infection. Nine (4·5%) TB ELISPOT
assays failed according to the manufacturer’s criteria. Five
patients had anergic PHA-induced IFN-g responses (four of
whom had CD4 < 15 cells/ml) and four individuals had high
background counts in the negative control well. Analysis of
the effect of low CD4 T cell counts on the performance of the
TB ELISPOT showed a weak correlation (Spearman’s
rho = 0·169, P = 0·017) between the CD4 T cell count and
the level of PHA-stimulated IFN-g production in HIV-1
patients. We identified 50 subjects with TB antigen-specific
immune responses, of whom 33 (66%) had CD4 T cell
counts < 300 cells/ml and nine (18%) had CD4 T cell counts
< 100 cells/ml. There was no significant correlation between
CD4 T cell count (Spearman’s rho =-0·036), CD8 T cell
count (Spearman’s rho = 0·103) and the magnitude of TB
antigen-specific IFN-g immune responses (Fig. 1). The sen-
sitivity of the TB ELISPOT assay for the diagnosis of active/
probable tuberculosis was 90·3% and was not affected by
CD4 T cell count (Table 2), although the limited number of
cases in HIV-1 patients with a CD4 T cell < 100 cells/ml
means that definitive conclusions about the performance of
this assay in very advanced HIV-1 associated immune sup-
pression remain to be determined. The specificity of the TB
ELISPOT for TB infection in this study was 100%. The assay
missed culture-positive tuberculosis in one individual (CD4
T cell count 17 cells/ml). A negative TB ELISPOT result was
obtained for one patient who had objective clinical and
radiological improvements, which was attributed to TB
treatment (CD4 T cell counts 447 cells/ml). No other symp-
tomatic patient with a negative TB ELISPOT assay developed
culture-positive tuberculosis or was started on empirical TB
treatment within 3 months of a negative test (Fig. 2). The
negative predictive value of the TB ELISPOT assay for the
diagnosis of TB infection in HIV patients who presented
with clinical or radiological features suggestive of this con-
dition was 98·2%. Follow-up of 20 patients with a positive
ELISPOT test result who were considered to have latent TB,
over a median period of 12 months, showed that two indi-
viduals developed active tuberculosis 3 and 10 months later;
however, large prospective studies are needed to define the
risk and positive predictive value of the TB ELISPOT test for
the development of active tuberculosis.
Individuals with active TB had significantly lower CD4 T
cells than patients with latent infection (median CD4 T cell
count 209 cells/ml versus 294 cells/ml, P = 0·043). There was
no significant difference in CD8 T cell counts between
patients with active tuberculosis and latent TB infection
(data not shown). The combined number of ESAT-6 and
CFP-10 IFN-g spot-forming cells to CD4 T cell count ratio
was significantly higher (median ratio 3·032 versus 0·694,
Table 2 . Performance of tuberculosis (TB) ELISPOT assay for diagnosis
of active tuberculosis in HIV-1 infected patients stratified by CD4 T cell
count.
Patient group No.
Sensitivity
(%)
Specificity
(%)
All HIV patients with
active/probable TB
30 90·3 100
CD4 T cell count < 300 cells/ml 22 95·4 100
CD4 T cell count < 200 cells/ml 14 92·9 100
CD4 T cell count < 100 cells/ml 8 87·5 100
2500
(a)
(b)
2000
1500
1000
500
0
0
0 500 1000 1500 2000 2500
200 400 600 800
Number of TB antigen-specific
T cells/10
6
PBMCs
2500
2000
1500
1000
500
0
Number of TB antigen-specific
cells/10
6
PBMCs
CD4 T cell count (cells/μl)
CD8 T cell count (cells/μl)
Fig. 1. Scatter plot of the magnitude of tuberculosis (TB)
antigen-specific immune responses against CD4 (a) and CD8 (b) T
cell counts. The sum of interferon (IFN)-g spots/10
6
peripheral blood
mononuclear cells (PBMC) in response to TB-specific peptide pools
early secretory antigen target-6 (ESAT-6) and culture filtrate
protein-10 (CFP-10) are plotted against CD4 (a) and CD8 (b) T cell
counts observed in each individual who had a positive test result.
Open circles represent patients with active/probable tuberculosis,
closed circles represent patients with latent TB infection. A positive
TB enzyme-linked immunospot assay was defined as follows: the sum
of the IFN-g spots to both peptide pools exceeds that seen in the
medium alone well by 25. In this set of experiments the number of
IFN-g spots seen in medium alone well in each individual was < 4
spots/10
6
PBMC.
S. A. Clark et al.
240
© 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 238–244
P = 003) in patients with active tuberculosis compared to
latent TB (Fig. 3a). Analysis of the combined number of
ESAT-6 and CFP-10 spots to CD8 T cell count ratio also
showed a significant difference (median ratio 1·012 versus
0·3690, P = 0·021) between patients with active and latent TB
(Fig. 3b). The positive predictive value, sensitivity and speci-
ficity for the diagnosis of active TB was 86%, 68% and 80%,
respectively, when the combined number of ESAT-6 and
CFP-10 spot count to CD4 T cell count ratio was > 1·5
(Fig. 3c). The positive predictive value was 79% when the
combined number of ESAT-6 and CFP-10 spot count to CD8
T cell count ratio was > 1·5; however, the sensitivity and
specificity was only 39% and 85%.
Eight patients did not return to have their TST read and
in one patient the TB ELISPOT assay did not work. There
was good agreement between the results of the TST and
TB ELISPOT (k=0·74, P < 0·001), with 89·06% (n = 63)
concordance. Discordant results were seen in seven patients,
four of whom had a positive TST and a negative TB
ELISPOT result, while three patients had a negative TST and
positive TB ELISPOT result. Two of three patients with a
positive TB ELISPOT test result and negative skin tuberculin
skin test had risk factors that are associated with reduced
TST sensitivity for TB infection (active TB disease, CD4 T
cell count 42 cells/ml). The reason for the discrepancy
between the TB ELISPOT and TST result in the final patient
with a history of latent TB is not known.
Discussion
We show that TB antigen-specific immune responses can be
detected in HIV-1 patients with reduced CD4 T cell counts
and that a combination of TB ELISPOT IFN-g spots and
CD4 T cell counts may distinguish active tuberculosis from
latent TB infection. The sensitivity of TST to diagnose active
tuberculosis in children and adults with HIV-1 infection is as
low as 36–40% [11], and the presence of skin anergy means
that false negative TST have been reported in 26–41% of
HIV-1 patients who are screened for latent TB infection
[12–14]. The sensitivity of the TB ELISPOT assay for the
diagnosis of active TB in immune-competent patients is
between 83 and 97%, with a specificity of 97–100% [15]. Our
data show similar figures for HIV patients, even in those with
CD4 T cell counts < 300 cells/ml, in whom the clinical and
radiological features of TB are modified by HIV-associated
immune deficiency. The sensitivity of the TB ELISPOT assay
for the diagnosis of active TB in HIV-1 patients was 90% in
a study in Zambia; however, there were no data available on
immune status of these patients [16]. Dheda and colleagues
showed that PHA-induced IFN-g secretion was not influ-
enced by low CD4 count in 29 patients with HIV-1 infection;
however, they did not assess TB antigen-specific immune
responses [17]. The TB ELISPOT and whole blood TB ELISA
IFN-g release assays (IGRA) gave comparable results in 74
HIV-1 infected patients when used to screen for latent TB
infection in a high-prevalence country [18]. The median
CD4 T cell count for HIV-1 infected patients was 392 cells/ml
and only 16 patients had a CD4 T cell count < 200 cells/ml, so
it was difficult to ascertain the influence of advanced HIV-
1-associated immune suppression on the performance of
these tests. Two studies have shown that whole blood TB
ELISA worked well in HIV-1 patients who had CD4 T cell
counts > 300 cells/ml in low-prevalence TB regions [19,20];
however, a high rate of assay failure due to low PHA
responses in HIV patients with CD4 T cell counts
< 100 cells/ml was noted. Similar findings have also been
noted by other groups assessing the clinical utility of whole
blood TB ELISA in other immune-deficient patients
[21–23]. The whole blood TB ELISA test was positive in only
two of 63 HIV-1 infected patients with a CD4 T cell count
< 200 cells/ml in a micronutrient trial that was conducted in
a TB endemic area [24], which raises concerns about the
performance of this particular test in immune-deficient
patients.
A combination of TB ELISPOT IFN-g spot frequency and
CD4 and CD8 T cell counts may have the potential to dis-
tinguish between active and latent TB infection in HIV-1-
infected patients. This agrees with a recent report from
Wilkinson and colleagues, who found that the sum of
Outcome
Tuberculosis
Bacterial pneumonia
RTI
OLI/NHL
Miscellaneous illness
24
36
22
21
47
Fig. 2. Clinical outcomes in all symptomatic patients who had a
tuberculosis (TB) enzyme-linked immunospot assay test. Clinical
outcomes for 154 symptomatic patients in this study were coded
using standardized criteria to define pneumonia, lower respiratory
tract infection, opportunistic infections and non-Hodgkin’s
lymphoma. The numbers of patients in each group were as follows:
tuberculosis (n = 36, 30 patients with active tuberculosis and six
individuals with latent TB infection) presumptive bacterial
pneumonia (n = 22), lower respiratory tract infection (n = 21),
opportunistic infections involving the lung [pneumocystis carinii
pneumonia (PCP), mycobacterium avium complex (MAC) and
non-Hodgkin lymphoma (NHL) (n = 47)], miscellaneous illnesses
(n = 24, including Gram-negative sepsis, pyelonephritis, inflammatory
lung disease, lung cancer, cerebrovascular accident, Hodgkin’s disease
acute illness now resolved). Five patients were lost to follow-up
(n = 3) or TB cultures are still outstanding (n = 1).
Immune responses to TB antigens in HIV
241
© 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 238–244
ESAT-6 and CFP-10 spots divided by CD4 T cell count could
distinguish between HIV-1 patients with pulmonary tuber-
culosis and newly diagnosed HIV-1 patients in South Africa
[25]. Further studies are needed to confirm this finding in
other HIV-1 cohorts and patient groups at risk of active
tuberculosis. In contrast to the African patients studied, 16%
of our patients were sputum smear-negative and 60% had
extrapulmonary disease, which suggests that this approach
may also work in patients whose TB disease is modified by
HIV-1-associated immune deficiency. Use of the ELISPOT
assay and CD4 T cell count in combination could accelerate
the diagnosis of TB infection in routine clinical practice
[26], and allow the commencement of anti-TB therapy
before the results of TB cultures are available in patients
who are believed to have symptoms/radiology consistent
with this disease [27]. We acknowledge that our sample size
is small and that larger prospective studies are needed to
confirm these preliminary findings. Other approaches to
distinguish between active disease and latent infection
include the identification of purified protein derivative
(PPD) [28–30] and TB [31,32] antigen-specific immune
responses in BAL or pleural fluid, even when no TB-specific
immune responses can be found in blood, and the use of
novel TB antigens which are specific for either active disease
or latent infection.
Agreement between the results of TST and IGRA range
between 53% and 94% in studies of immunocompetent
individuals screened for latent TB infection or studies in
contact tracing [19,21,23,33]. In this study, discordance
18
(a)
16
14
12
10
8
6
4
2
0
Active TB Latent TB
Ratio of IFN-γ SFC to CD4 T cells
(c)
18
16
14
12
10
8
6
4
2
0
0 200
CD4 T cell count (cells/μl)
400 600 800
Ratio of IFN-γ SFC to CD4 T cells
(b)
8
6
4
2
0
Active TB Latent TB
Ratio of IFN-γ SFC to CD8 T cells
Fig. 3. The ratio of the combined number of early secretory antigen
target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) interferon
(IFN)-g spot-forming cells (SFC) per 10
6
peripheral blood
mononuclear cells (PBMC) to CD4 and CD8 T cell count has the
potential to distinguish active tuberculosis (TB) from latent TB
infection in this patient cohort. (a) Comparison of the combined
number of ESAT-6 and CFP-10 IFN-g SFC to CD4 T cell count ratio
in patients with active TB (median ratio 3·032) and latent TB
infection (median ratio 0·694) was found to be statistically significant
(P = 0·003). Horizontal lines indicate median values. While the ratio
of the number ESAT-6 IFN-g spots to CD4 T cell counts was
significantly higher in patients with active TB compared to latent TB
(median ratio 1·189 versus 0·177, P = 0·018), no significant difference
was observed for CFP-10 (median ratio 1·095 versus 0·319, P = 0·084).
(b) Comparison of the combined number of ESAT-6 and CFP-10
IFN-g SFC to CD8 T cell count ratio in patients with active TB
(median ratio 1·015) and latent TB infection (median ratio 0·369) was
found to be statistically significant (P = 0·021). Horizontal lines
indicate median values. No significant difference was observed in
patients with active TB compared to latent TB in the number of
ESAT-6 or CFP-10 IFN-g spots to CD8 T cell count ratio. (c) Scatter
plot of the combined number of ESAT-6 and CFP-10 IFN-g SFC to
CD4 T cell count ratio plotted against CD4 T cell count. Open circles
represent patients with active/probable TB infection, closed circles
represent patients with latent TB infection. The dotted line represents
a ratio of 1·5.
S. A. Clark et al.
242
© 2007 British Society for Immunology, Clinical and Experimental Immunology, 150: 238–244
between the results of TST and TB ELISPOT occurred in
11% of patients tested, which agrees with previous reports
[34,35]. A history of previous bacille Calmette–Guérin
(BCG) vaccination, exposure to environmental mycobacte-
ria, recent exposure to TB or previous TB treatment may
account for some TST
+
IGRA
test results. Reduced sensitiv-
ity of IGRA compared with TST has been described [36], and
may be a feature of individuals with a history of remote TB
exposure [33,37], as was observed in three of four patients in
this study. Positive TST responses may persist, whereas TB
ELISPOT assays may revert from positive to negative either
spontaneously [38–40] or, in some cases, after TB therapy
[41–45]. Reversion of IGRA test results may also be influ-
enced by precision of assays used and definition of positive
test results [20,35]. Conditions associated with reduced TST
sensitivity for TB infection (active TB disease and a CD4 T
cell count < 100 cells/ml) may explain the positive TB
ELISPOT responses and negative TST observed. The concen-
tration and type of tuberculin preparation may influence the
rate of discordant tests results and a criticism of this and
other studies [18,25] is that the PPD dose employed may be
lower than recommended for clinical diagnosis of TB [9],
resulting in potential bias for false negative TST skin test
results.
Further studies are required to define the CD4 threshold
where the test performance of this assay declines, in particu-
lar whether the sensitivity of this assay will decline with CD4
T cell counts < 50 cells/ml. Prospective studies are needed
to define what proportion of HIV patients who adhere to
TB chemotherapy or prophylaxis will have undetectable
TB antigen-specific immune responses in blood at the
end of treatment and whether TB immune reconstitution
syndrome (IRIS) is associated with persistent ESAT-6 or
CFP-10-dependent T cell IFN-g secretion [46].
In conclusion, we find the M. tuberculosis-specific TB
ELISPOT test can detect TB antigen-specific immune
responses in HIV-1 patients with low CD4 T cell counts. This
assay is a useful test to screen for TB infection even in HIV-1
patients with low CD4 T cell counts, and can be used in
routine clinical practice. A combination of TB ELISPOT spot
number and CD4 T cell count may have the potential to
distinguish between active and latent TB infection.
Acknowledgements
We would like to thank the following for their help with
this project: Sr Sheena Basnayake, TB specialist nurse at the
Chelsea & Westminster NHS Foundation Trust, Dr E. Low,
SpR HIV/GUM Directorate Chelsea & Westminster NHS
Trust and Dr C. Cohen, consultant HIV/GUM West
London Sexual Health Clinic, Charing Cross Hospital,
Chelsea & Westminster Foundation NHS Trust. We are
grateful to the immunology laboratory staff for their help
in performing CD4 T cell counts and HIV-1 viral load
assays. Finally, we want to thank the patients who took part
in this study. P. Kelleher has received grant support from
the St Stephens AIDS Trust, London, UK to conduct this
study. P. Kelleher and A. Pozniak acknowledge Oxford
Immunotec, Oxford, UK for their generous supply of the
T-SPOT TB assay.
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    • "A decrease in polyfunctional (IFNγ+ IL2+ and TNFα +) antigen specific cells has also been noted in the airways of patients with HIV [12][13][14]. Such findings strengthen the hypothesis that HIV reduces the number and function of Mtb specific CD4 + T cells [4,5,[15][16][17][18][19]. Other studies have demonstrated higher numbers of Mtb antigen specific CD4+ T cells in coinfected patients despite lower total CD4 + T cell counts [20][21][22]. The dynamics of the immune system, particularly the function of CD4 + T cells after commencement of treatment for either HIV or TB are quite complex. "
    [Show abstract] [Hide abstract] ABSTRACT: Characterizing perturbations in the immune response to tuberculosis in HIV can develop insights into the pathogenesis of coinfection. HIV+TB+ and TB monoinfected (TB+) subjects recruited from clinics in Bamako prior to initiation of TB treatment were evaluated at time-points following initiation of therapy. Flow cytometry assessed CD4+/CD8+Tcell subsets and activation markers CD38/HLA-DR. Antigen specific responses to TB proteins were assessed by intracellular cytokine detection and proliferation. HIV+TB+ subjects had significantly higher markers of immune activation in the CD4+ and CD8+Tcells compared to TB+ subjects. HIV+TB+ had lower numbers of TB-specific CD4+Tcells at baseline. Plasma IFNγ levels were similar between HIV+TB+ and TB+ subjects. No differences were observed in in-vitro proliferative capacity to TB antigens between HIV+TB+ and TB+ subjects. Subjects with HIV+TB+ coinfection demonstrate in vivo expansion of TB-specific CD4+Tcells. Immunodeficiency associated with CD4+Tcell depletion may be less significant compared to immunosuppression associated with HIV viremia or untreated TB infection. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Apr 2015
    • "In all of these assays, a clear understanding of which antigens from MTB elicit immune responses is direly needed. Recent studies have focused on the identification of new antigens262728293031. These studies have varied from in silico approaches,[31]to gene expression approaches[29]as well as MHC-specific methods[26,27,30]. "
    Article · Jan 2015 · BMC Infectious Diseases
    • "Our study where none developed active TB regardless of preventive therapy is in contrast to the Swiss study were 6.5% of HIV-patients not treated for LTBI acquired active TB during the follow-up of 52 months [12]. A PPV of 8-10% for progress to active TB infection in IGRA positive HIV infected patients within a two years period has also been documented from other TB low-endemic countries [29,30]. The fact that CD4 counts were lower and fewer patients were on ART in these studies compared to our study could partly explain these conflicting data. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Interferon-gamma release assays (IGRA) serve as immunodiagnostics of tuberculosis (TB) infection to identify individuals with latent TB infection (LTBI) eligible for preventive anti-TB therapy. In this longitudinal study of HIV-infected LTBI patients we have observed for possible progression to active TB as well as evaluated repeated IGRA testing in a TB low-endemic setting. Methods: QuantiFERON TB-Gold In-tube® assay (QFT), TB-SPOT.TB® (TSPOT) and tuberculin skin test (TST) were performed on 298 HIV-patients recruited from seven out-patient clinics in Norway. Patients with active TB, LTBI and negative IGRA were followed with repeat QFTs and clinical evaluation over a period of 24 months. Results: Seven HIV-patients (median CD4 count 270; IQR 50¿340) were diagnosed with active TB at inclusion, all IGRA positive. Sixty-four (21%) HIV-patients (median CD4 count 471; IQR 342+-638) were diagnosed with LTBI and of these 39 (61%) received TB preventive treatment. Neither treated nor untreated HIV-infected LTBI patients developed active TB during the 24 months. At baseline, the median interferon-gamma (INF-gamma) level measured by QFT was 3.48 IU/ml (IQR 0.94 +- 8.91 IU/ml) for treated LTBI compared to 1.13 IU/ml (IQR 0.47 +- 4.25 IU/ml) for untreated LTBI patients (p=0.029). The QFT reversion rates were 75% for active TB, 23% for treated LTBI and 44% for untreated LTBI, whereas the conversion rate for the non-TB group was 7% despite no new TB exposure. There was no significant difference in the trend of INF-gamma levels over time between treated and untreated LTBI patients. Conclusion: The prevalence of LTBI is high among HIV-patients, but the risk of developing active TB seems to be low in patients with high CD4 counts in this TB low-endemic setting. In several patients, especially with baseline IFN-gamma levels close to cut-offs, the QFT tests reverted to negative independent of preventive anti-TB treatment indicating possibly false positive tests. This highlights the importance of defining reliable cut-offs for immunodiagnostic tests and deferring preventive therapy in selected patients. Randomized studies with longer follow-up time are needed to identify HIV-patients that would benefit from LTBI treatment in a TB low-endemic setting.
    Full-text · Article · Dec 2014
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