Indian Journal of Experimental Biology
Vol. 47, June 2009, pp. 463-468
Evaluation of whole blood IFNγ test using PPD and recombinant antigen
challenge for diagnosis of pulmonary and extra-pulmonary tuberculosis
Yatiraj Kalantri, Nanda Hemvani & D S Chitnis*
Department of Microbiology and Immunology, Choithram Hospital and Research Centre, Indore 452 014, India
Received 23 January 2009
Quantiferon TB gold (QFT-G) with recombinant antigen cocktail is well evaluated for diagnosis of pulmonary
tuberculosis (PTB). However, diagnosis of extra-pulmonary tuberculosis (EPTB) is more difficult due to limitations of
conventional techniques. This study compares recombinant antigens based QFT-G and low cost PPD based interferon test
for the diagnosis of PTB and EPTB. IFNγ release, with recombinant antigens and PPD, was assayed by ELISA from 140
cases of EPTB, 100 cases of PTB along with acid fast bacillus (AFB) detection, AFB culture on LJ and MGIT BACTEC.
Sensitivity and specificity for QFT-G recombinant antigens was 84.29% and 96%, while for PPD based interferon was 70%
and 84% for EPTB group. The sensitivity was far superior to AFB smear and culture for both the antigens. Nine samples
were identified as non-tubercular mycobacteria (NTM) in the EPTB group and all were negative for QFT-G, but six of them
were positive for PPD based test. Results of the study show that QFT-G using recombinant antigen is sensitive and specific
for both PTB and EPTB diagnosis. The PPD based test is economic and offers comparable performance for PTB and EPTB
diagnosis and also useful for diagnosis of NTM.
Keywords: CFP-10, ESAT-6, Extra-pulmonary tuberculosis, IFNγ, Non-tubercular mycobacteria, PPD, TB
One-third of the world’s population is infected with
Mycobacterium tuberculosis1, and tuberculosis (TB)
remains a major worldwide cause of morbidity and
mortality. It is estimated that extra-pulmonary
tuberculosis (EPTB) constitutes 20% of all cases of
tuberculosis in immunocompetent patients and
accounts for more than 50% of cases in HIV positive
individuals2-10.
Clinical presentation together with radiological and
laboratory findings make the diagnosis of pulmonary
tuberculosis (PTB) easier. However, the diagnosis of
EPTB is usually more challenging as radiographic
analysis is often not conclusive, the site of infection
relatively inaccessible and the site may be fewer
bacilli can cause considerably greater harm because of
the types of organs infected. Invasive procedures are
therefore often needed for confirmation of tentative
diagnosis11.
Detection of cell-mediated immunity by tuberculin
skin test (TST) has for nearly a century been used as a
surrogate marker for infection with Mycobacterium
tuberculosis, both as a diagnostic aid and in
epidemiological studies. The main drawback with the
________________
*Correspondent author
Telehone: 91-0731-2362491-99; Fax : 91-0731-2470068
E-mail : [email protected]
clinical use of TST is lack of specificity due to crossreactivity with other mycobacteria such as
Mycobacterium bovis12,13. High endemicity and mass
BCG vaccination are the other limitations.
A simple diagnostic test based on whole blood was
used in an attempt to overcome some of the
limitations of TST14-18. The Quantiferon TB gold in
tube test (Cellestis, Australia) measures interferon
gamma (IFNγ) production after in vitro stimulation of
whole blood cells with Mycobacterium tuberculosis
specific antigen cocktail containing early secreted
antigenic target 6 (ESAT-6), culture filtrate protein 10
(CFP-10) and TB7.7 (p4). ESAT-6 and CFP-10
antigens are encoded by genes located within the
region of difference 1 (RD1) segment of the M.
tuberculosis genome and are more specific because
they are not shared with any of the BCG vaccine
strains and most of the non-tuberculous mycobacteria
(NTM). TB7.7 (Rv2654) is termed as peptide-4 which
is specific for M. tuberculosis complex. Several
studies have documented high sensitivity and
specificity for detection of active PTB infection14-18,
but the data is scanty for the diagnosis of EPTB19, 20.
The aim of the present study was to evaluate the
response of lymphocytes from EPTB and PTB cases
to M. tuberculosis specific antigens cocktail mixture
(ESAT-6, CFP-10, TB7.7) the tube using the
INDIAN J EXP BIOL, JUNE 2009
464
Quantiferon TB test and its comparison to PPD
antigen challenge. The data was analyzed in the
background of AFB smear and culture findings for the
cases.
Materials and Methods
Specimens for study
Cases of EPTB(140) referred to Choithram
Hospital and Research Center, (CHRC), Indore, India
were included in the study. Males (98) and females
(42) between the age group of 18-50 years. Four
groups were involved in the study–healthy volunteers,
EPTB cases, bacteriologically confirmed PTB cases
and non tubercular lung infection cases (Table 1). The
study, conducted from January 2007 to July 2008,
was approved by the Research and Ethical Committee
of CHRC, Indore, India. The Ethical Committee has
guidelines based on Helsinki declaration.
Healthy volunteers group—Healthy voluntary
blood donors from Indore city and the adjoining area
who came for blood donation at the blood bank of
CHRC were included as normal population.
Volunteers (50) were in the age group of 22-40 years
and neither had no history of any notable infection in
the past 2 years, nor had symptomatic tuberculosis in
the lifetime.
Table 1—Categories of subjects included in the study
Category
Healthy volunteers (50)
Extra-pulmonary TB (140)
TB lymphadenitis (55)
TB pleuritis (34)
Bone TB (20)
Genitourinary TB (14)
Abdominal TB (8)
TB meningitis (5)
Miliary TB (3)
Occular TB (1)
Bacteriological confirmed PTB cases
(100)
Non-tubercular lung infection cases (50)
Malignancies (15)
COPD (5)
Pneumonia (8)
Sarcoidosis (6)
Bronchiectasis (8)
Pancretitis (4)
Pulmonary embolism (4)
Male Female
Age
(yr)
35
98
15
42
22-40
21-50
76
24
24-45
34
16
23-47
Number of volunteers in each category is given in parenthesis
PTB group—100 cases of PTB were included in the
study. Criteria for inclusion were–active PTB infection,
based on clinical, radiological and sputum AFB smear
positivity. All the cases had cough (>3 week duration)
with expectoration, weight loss, fever, tiredness, loss of
appetite, breathlessness, and chest pain. Chest X-rays
showed a classical pattern of upper lobe
infiltrate/bilateral infiltrate/cavitation with or without
pulmonary fibrosis and shrinkage.
Non-tubercular lung group—Another control group
of 50 cases had lung infection wherein tuberculosis
was ruled out on the basis of AFB smear negativity in
three consecutive samples and subsequently AFB
culture negativity. Their diagnosis is mentioned in
Table 1.
EPTB group— 149 cases of EPTB were included in
the study. Diagnosis of EPTB was made on the basis of
clinical manifestations consistent with TB and
positivity for one or more of the following parameters–
AFB microscopy, AFB culture, histological or biopsy
findings and raised ADA, lymphocytes and proteins
and response to anti-TB drugs.
Specimen collection and processing
Specimens were decontaminated and concentrated
using NaOH-NALC method21. Centrifuged deposit
with about 2 mL supernatant mixed by vortexing
formed the concentrate. An aliquot of 0.5 mL of the
concentrate was inoculated over Lowenstein Jensen
(LJ) slant and MGIT BACTEC tube. The LJ slant were
incubated at 37°C for 4-8 weeks and observed weekly.
The MGIT tube was observed on alternate days using
micro MGIT fluorescent reader (BD BBL, USA).
Another aliquot of 0.5 mL concentrate was
recentrifuged in microtubes at 6000 rpm for 5 min and
the deposit was used for AFB smear. The smear stained
with Auramin “O” (Hi-Media, India) was seen under
fluorescent microscope. Identification of nontubercular mycobacteria (NTM) was based on
biochemical tests [niacin, catalase, para-nitro benzoic
acid (PNB)]. The blood samples were collected for T
cell Interferon gamma assay.
T cell interferon-γ assay
Activation of T cells in tuberculosis cases—Aliquots
(1mL) of heparanized whole blood were incubated
with (i) recombinant antigen [ESAT-6, CFP-10,
TB7.7(p4)], (ii) PPD (10 TU-PPD, 2μg/mL) (Span
Diagnostics, India) and (iii) nil control. Following 16 to
24 hr incubation plasma was removed and the amount
of IFN-gamma was quantified by ELISA.
KALANTRI et al.: WHOLE BLOOD IFNγ TEST FOR TB DIAGNOSIS
Measurement of IFN-gamma levels—Interferon
gamma levels were measured by enzyme linked
immunosorbent assay (ELISA) method using
Quantiferon TB Gold kit as per the protocol suggested
by the manufacturer. The conjugate (anti-human IFNγ
HRP) dilutions were prepared with diluent (bovine
casein, normal mouse serum) and 50 μL were added to
the wells coated with monoclonal mouse anti-human
IFNγ. Test plasma (50 μL) and standards were added to
the wells and incubated for 120 min at room
temperature. Wells were washed for six times. Enzyme
substrate solution (tetra-methly-benzidine) (100 μL)
was added to the wells and incubated for 30 min. at
room temperature. Stop solution (H2SO4) (50 μL) was
added and OD measured at 450 nm with 620 nm
reference filter. Standard curve was constructed as per
the manufacturers instructions to quantify the IFNγ
concentration (IU/mL) for each of the test plasma
samples. Cutoff value for a positive response was set as
0.35 IU/mL for recombinant antigens (ESAT-6, CFP10 and TB7.7(p4)) as per the manufacturer instructions.
The cut of 0.5 IU/mL for PPD was obtained by adding
mean ± 2 SD values from healthy volunteers group.
Statistical analysis—Data was analysed using
diagnostic test 2 × 2 contingency tables to calculate
sensitivity, specificity, positive predictive value (PPV),
negative predictive value (NPV) and likelihood ratios
(LR) using StatsDirect statistical software version
2.7.2, UK. Differences between various diagnostic
methods were measured using McNemar X2 test (P
values <0.05 were considered significant).
Results
Out of the 140 cases of EPTB, AFB staining was
positive in only 42 cases whereas 54 samples were
found culture positive among the EPTB Group. In the
465
EPTB group 9 cases were found to be NTM based on
biochemical identification of the isolates. All 9 NTM
isolates were rapid growers–M. fortuitum (8) and M.
chelonae (1). Positivity for various tests and NTM
isolation among EPTB cases are shown in Table 2.
In PTB group among the 4 parameters the highest
sensitivity was recorded for T-cell IFNγ (96%) using
recombinant antigens followed by, T-cell IFNγ (82%)
using PPD, culture by LJ and MGIT-BACTEC (84%)
and AFB by fluorescent microscopy (74%). Sensitivity,
specificity, PPV, NPV and likelihood ratio values for
different tests for samples from PTB, healthy
volunteers and non-TB lung infection samples are
shown in Table 3.
Among the 4 parameters in EPTB group the highest
sensitivity was recorded for T-cell IFNγ (84.29%)
using recombinant antigens (ESAT-6, CFP-10 and
TB7.7). In the EPTB group T-cell IFNγ using
recombinant antigens (ESAT-6, CFP-10 and TB7.7)
had high sensitivity than AFB smear, culture by LJ
media and MGIT-BACTEC (95% CI, P<0.0001,
McNemar Test). Similarly T-cell IFNγ using PPD
antigen had high sensitivity than AFB smear, culture
by LJ media and MGIT-BACTEC (95% CI, P<0.0001,
McNemar Test). Sensitivity, specificities and
likelihood ratio values were high for T-cell IFNγ using
recombinant antigens than for PPD but the differences
by two-sided McNemar test were not significant (95%
CI, P=0.31). Data for both EPTB group, healthy
volunteers and non-TB lung infection group with their
sensitivity, specificity, NPV, PPV and likelihood ratio
for different parameters are given in Table 4.
Among the 9 cases infected with NTM in the
EPTB group, 6 were found positive by PPD–T-cell
IFNγ assay, where as all 9 NTM were found negative
by recombinant T-cell IFNγ assay.
Table 2—Positivity for various parameters and NTM isolation among EPTB cases
EPTB Category
TB-lymphadenitis (55)
TB pleuritis (34)
Bone TB (20)
Genitourinary TB (12)
Abdominal TB (10)
TB meningitis (5)
Milliary TB (3)
Ocular TB (1)
Total No. of EPTB cases (140)
AFB smear Culture by LJ and No. of NTM T-Cell IFNγ using combined T-Cell IFNγ using
positive case MGIT-BACTEC
isolated
antigens (ESAT-6, CFP-10
PPD antigen
positive cases
and TB7.7) positive cases
positive cases
22
9
6
3
2
42
24
11
8
5
3
2
1
54
Number of volunteers in each category is given in parenthesis
4
1
1
2
1
9
50
30
17
8
6
3
3
1
118
44
25
14
6
3
2
3
1
98
INDIAN J EXP BIOL, JUNE 2009
466
Table 3—Sensitivity, specificity, PPV, NPV and LR values for different parameters for samples from PTB, healthy volunteer group and
non-tuberculous lung infection samples
Parameters
Healthy
volunteer
group (50)
AFB smear
Culture by LJ and MGITBACTEC
T-cell- IFNγ (ESAT-6,
CFP-10 and TB 7.7)
T-cell- IFNγ (PPD)
Positive cases
in non-TB
group (50)
Positive cases in
PTB group (100)
Sensitivity Specificity
(%)
(%)
PPV
(%)
NPV
(%)
LR
Nil
Nil
Nil
Nil
74
84
74
84
100
100
100
100
65.78
75.75
10.35-infinity
11.7-infinity
2
2
96
96
96.15
97.95
95.59
24.93
8
8
82
82
84
91.11
73.52
5.12
PPV–positive predictive value; NPV–negative predictive value; LR–likelihood ratio
Number of volunteers in each category is given in parenthesis
Table 4—Sensitivity, specificity, PPV, NPV and LR values for different parameters in EPTB, healthy volunteer group and
non-tuberculous lung infection samples
Parameters
AFB smear
Culture by LJ and
MGIT-BACTEC
T-cell- IFNγ
(ESAT-6, CFP-10
and TB 7.7)
T-cell- IFNγ (PPD)
Healthy
volunteer
group (50)
Nil
Positive cases Positive cases
in non-TB
in EPTB
group (50) group (140)
Nil
42
Sensitivity
(%)
Specificity
(%)
PPV
(%)
NPV
(%)
LR
30
100
100
33.78
Nil
Nil
54
38.57
100
100
36.6
2
2
118
84.29
96
98.33
68.57
4.18infinity
5.38infinity
21
8
8
98
70
84
92.45
50
4.37
PPV–positive predictive value; NPV–negative predictive value; LR–likelihood ratio
Number of volunteers in each category is given in parenthesis
Discussion
Rapid diagnosis and treatment of tuberculosis is
crucial to reduce morbidity and mortality from
untreated TB. EPTB accounts for approximately one
third of all cases of tuberculosis22,23. Conventional
methods have limitations for the diagnosis of EPTB.
The smear for AFB was negative for 98 of the 140
samples from EPTB cases at our end and pointed out
the low diagnostic yield for the parameter. The culture
test requires 50 to 1,000 bacilli/mL in the sample.
Yield of AFB cultures by LJ and MGIT-BACTEC
was 38.57%. Prescription of ofloxacin or
ciprofloxacin as empirical antimicrobial therapy for
respiratory tract and other infections in our area
appeared to be one of the limiting factors for culture.
Tuberculin skin test (TST) has been used for
almost a century to support the diagnosis of TB
infection24. However, TST has limitations such as low
specificity among BCG vaccinated individuals or
sensitized with NTM. A more specific diagnostic
assay should preferably be based on antigens that are
present exclusively in MTB but not in BCG. Because
of advances in molecular biology and genomics, a
major breakthrough in recent years has been the
development of in vitro assays that measure T-cell
release of interferon γ in response to stimulation with
antigens such as ESAT-6 and CFP-10. ESAT-6 and
CFP-10 antigens are encoded by genes located within
the region of difference 1 (RD1) segment of the M.
tuberculosis genome and are more specific because
they are not shared with any of the BCG vaccine
strains and from most of the NTM species. TB 7.7
(peptide 4) is added to kit by manufacturer as it is
specific to M. tuberculosis complex and will detect
some sites which are missed by ESAT-6 and CFP-10.
These antigens are considered to be potential
candidates for immunodiagnosis of tuberculosis.
There have been several reports to support the
utility of T-cell release of IFNγ for the diagnosis of
acute PTB14-18. However, the reports over the
evaluation of TB Quantiferon test for the diagnosis of
EPTB have been fever19, 20.
In the present series the PTB group was included
for comparing the diagnostic utility of the test for
EPTB cases. Sensitivity for T-cell interferon test
using specific antigen combination (ESAT-6, CFP-10
and TB 7.7) was 84.29% for EPTB population while
96% for PTB population. Specificity for both groups
KALANTRI et al.: WHOLE BLOOD IFNγ TEST FOR TB DIAGNOSIS
was 96% and 96.15%, respectively. Thus, the
diagnostic efficiency of the test appears equal for both
PTB and EPTB cases. Similar observation was
reported by Nishimura et al.19 from Japan. Ravan et
al.17 also reported greater sensitivity of the test for
EPTB cases as compared to PTB cases. However, the
population size included in the study was much
smaller.
The notable observations in the present study was
that sensitivity and specificity of the T-cell interferon
release following PPD challenge appears lower than
with specific combination antigen (ESAT-6, CFP-10,
TB 7.7) challenge in Quantiferon test. However, the
differences between both the challenges were not
found to be statistically significant (P=0.31). The
observation has immense financial bearing since the
cost of locally available PPD antigen drastically
reduced the reagent cost in comparison to imported
Quantiferon kit (Rs 250 vis-a-vis Rs 1000). Brock et
al.16 reported similar sensitivity for PPD antigen and
ESAT-6 or CFP-10 antigen. However, PPD antigen
had shown positivtiy of 47% among BCG vaccinated
group with no active TB infection in Danish
population. In present study, only 8 of the 50 (16%)
cases in healthy volunteers had shown positivity for
PPD antigen based interferon rise. Eight of the 50
(16%) non-TB lung infection cases had shown
positive for PPD antigen based interferon rise. It was
not possible to ascertain whether these non-TB cases
had latent TB infection.
The most striking observation was the elevation of
T-cell interferon following PPD challenge among 6 of
9 cases infected with NTM, while none of the NTM
cases showed rise in interferon following ESAT-6,
CFP-10 and TB 7.7 antigen challenge. Thus, the
Quantiferon test confirmed the specificity of the
antigen combination for detecting exclusively M.
tuberculosis, but will be missing interferon caused by
NTM. Our earlier study has pointed out prevalence of
NTM among EPTB cases to be 38%25.
Based on present observation, it is expressed that the
PPD based challenge to mononuclear blood cells and
measurement of interferon can be a reasonable test for
the diagnosis of EPTB caused by both M. tuberculosis
and NTM and can prove much more economical
compared to Quantiferon TB Gold test. Multicentric
trials for the PPD based interferon test are suggested in
developing countries before its final recommendations.
Diagnostic utility of the test also needs to be evaluated
among HIV or immunocompromised groups.
467
Acknowledgement
Financial support from ICMR (Indian Council of
Medical Research) New Delhi, India and
infrastructural support by Management, Choithram
Hospital and Research Center, Indore, India are
gratefully acknowledged.
References
1 Arend S M, Engelhard A C, Groot G, de Boer K, Anderson P,
Ottenhoff T H M & van Dissel J T, Tuberculin skin testing
compared with T-cell responses to Mycobacterium
tuberculosis-specific and nonspecific antigens for detection of
latent infection in persons with recent tuberculosis contact,
Clin Diagn Lab Immunol, 8 (2001) 1089.
2 Fanning A, Tuberculosis: 6, Extrapulmonary disease, CMAJ,
160 (1999) 1597.
3 Mohan A & Sharma S K, Epidemiology in Tuberculosis
(Jaypee Brothers Medical Publishers, New Delhi) 2001, 29.
4 Raviglione M C, Narain J P & Kochi A, HIV-associated
tuberculosis in developing countries: Clinical features,
diagnosis and treatment, Bull World Health Organ, 70 (1992)
515.
5 Haas D W & Des Prez R M, Tuberculosis and acquired
immunodeficiency syndrome: A historical perspective on
recent developments, Am J Med, 96 (1994) 439.
6 Shafer R W, Kim D S, Weiss J P & Quale J M,
Extrapulmonary tuberculosis in patients with human
immunodeficiency virus infection, Medicine (Baltimore), 70
(1991) 384.
7 Lado Lado F L, Barrio Gomez E, Carballo Arceo E, Cabarcos
Ortiz de Barron A, Clinical presentation of tuberculosis and
the degree of immunodeficiency in patients with HIV
infection, Scand J Infect Dis, 31 (1999) 387.
8 Lee M P, Chan J W, Ng K K & Li P C, Clinical manifestations
of tuberculosis in HIV-infected patients, Respirology, 5 (2000)
423.
9 Poprawski D, Pitisuttitum P & Tansuphasawadikul S, Clinical
presentations and outcomes of TB among HIV-positive
patients, Southeast Asian J Trop Med Public Health, 31 (2000)
140.
10 Sharma S K & Mohan A, Extrapulmonary tuberculosis,
Indian J Med Res, 160 (2004) 316.
11 American Thoracic Society and the Centers for Disease
Control and Prevention, Diagnostic standards and
classification of tuberculosis in adults and children, Am J
Respir Crit Care Med, 161 (2000) 1376.
12 Hubener R E, Schein M F & Bass J B, The tuberculin skin test,
Clin Infect Dis, 17 (1993) 968.
13 Felten M K & van-der-Merwe CA, Random variation in
tuberculin sensitivity in schoolchildren. Serial skin testing
before and after preventive treatment for tuerculosis, Am Rev
Respir Dis, 140 (1989) 1001.
14 Streeton J A, Dessem N & Jones S L, QuantiFERON-TB: A
new in vitro test for tuberculosis and atypical mycobacterial
infection, Am J Respir Crit Care, 151 (1995) A511.
15 Desem N & Jones S L, Development of a human gamma
interferon enzyme immunoassay and comparison with
tuberculin skin testing for detection of Mycobacterium
tuberculosis infection, Clin Diagn Lab Immunol, 5 (1998) 531.
16 Brock I, Munk M E, Kok-Jensen A & Anderson P,
468
17
18
19
20
INDIAN J EXP BIOL, JUNE 2009
Performance of whole blood IFNγ test for tuberculosis
diagnosis based on PPD or the specific antigens ESAT-6 and
CFP-10, Int J Tuberc Lung Dis, 5 (2000) 462.
Raven P, Munk M E, Anderson A B, Lundgren B, Lundgren J
D, Nielsen L N, Kok-Jensen A, Anderson P & Weldingh K,
Prospective evaluation of Whole-Blood test using
Mycobacterium tuberculosis- specific antigens ESAT-6 and
CFP-10 for Diagnosis of Tuberculosis, Clin Diagn Lab
Immunol, 12 (2005) 491.
Brock I, Weldingh K, Lillebaek T, Follmann F & Anderson P,
Comparison of tuberculin skin test and new specific Blood test
in Tuberculosis contacts, Am J Res Crit Care Med, 170 (2004)
65.
Nishimura T, Hasegawa N, Mori M, Takebayashi T, Harada
N, Higuchi K, Tasaka S & Ishizaka A, Accuracy of an
interferon-γ release assay to detect active pulmonary and extrapulmonary tuberculosis Int J Tuberc Lung Dis, 12 (2008) 269.
Song K H, Jeon J H, Park W B, Kim S H, Park K U, Kim N J,
Oh M D, Kim H B & Choe K W,Usefulness of the whole-
21
22
23
24
25
blood interferon-gamma release assay for diagnosis of
extrapulmonary tuberculosis, Diagn Microbiol Infect Dis, 63
(2008) 182.
Kubica G P, Gross W M, Hawkins J E, Sommers H M, Vestal
A L & Wayne L G, Laboratory services for mycobacterial
diseases, Am Rev Respir Dis, 112 (1975) 783.
Weir M R & Thornton G F, Extrapulmonary tuberculosis:
Experience of a community hospital and review of the
literature, Am J med, 79 (1985) 467.
Dolberg O F, Sclaeffer F, Greene V N & Alkan M L
Extrapulmonary Tuberculosis is an immigrant society: Clinical
and demographic aspects of 92 cases, Rev Infect Dis, 13 (1991)
177.
American Thoracic Society, Targeted tuberculin testing and
treatment of latent tuberculosis infection, MMWR, 49 (2000) 1.
Hemvani N, Chitnis D S, Bhatia G C & Soni N,
Bacteriological study on pulmonary and extra-pulomnary cases
of tuberculosis from central India, Indian J Med Sci, 54 (2000)
188.