Poor performance of serological tests in the diagnosis of pulmonary tuberculosis: evidence from a contact tracing field study.

Abstract

Delayed or missed diagnosis of TB continues to fuel the global TB epidemic, especially in resource limited settings. Use of serology for the diagnosis of tuberculosis, commonly used in India, is another factor. In the present study a commercially available serodiagnostic assay was assessed for its diagnostic value in combination with smear, culture and clinical manifestations.
A total of 2300 subjects were recruited for the study, but 1041 subjects were excluded for various reasons. Thus 1259 subjects were included in the study of which 470 were pulmonary tuberculosis cases (440 of 470 were culture-positive) and 789 were their asymptomatic contacts. A house-to-house survey method was used. Blood samples were tested for IgM, IgA, and IgG antibodies using the Pathozyme Myco M (IgM), Myco A (IgA) and Myco G (IgG) enzyme immunoassay (EIA). Out of 470 PTB cases, BCG scar was positive in 82.34%. The Mantoux test and smear positivity rates in PTB cases were 94.3% (430/456), and 65.32% (307/470), respectively. Among the asymptomatic contacts, BCG scar was positive in 95.3% and Mantoux test was positive in 80.66% (442/548) contacts. No contact was found falsely smear positive. The sensitivity of IgM, IgA, and IgG EIA tests was 48.7%, 25.7% and 24.4%, respectively, while the specificity was 71.5%, 80.5%, 76.6%, respectively. Performance of EIAs was not affected by the previous BCG vaccination. However, prior BCG vaccination was statistically significantly (p = 0.005) associated with Mantoux test positivity in PTB cases but not in contacts (p = 0.127). The agreement between serology and Mantoux test was not significant.
The commercial serological test evaluated showed poor sensitivity and specificity and suggests no utility for detection of pulmonary tuberculosis.

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Poor Performance of Serological Tests in the Diagnosis of
Pulmonary Tuberculosis: Evidence from a Contact
Tracing Field Study
Sarman Singh1*, Jitendra Singh1., Sandeep Kumar1., Krishnamoorthy Gopinath1, Veena Balooni1,
Niti Singh2, Kalaivani Mani3
1Clinical Microbiology Division, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India, 2Lala Ram Sarup Institute of Tuberculosis
and Respiratory Diseases, New Delhi, India, 3Department of Biostatics, All India Institute of Medical Sciences, New Delhi, India
Abstract
Background: Delayed or missed diagnosis of TB continues to fuel the global TB epidemic, especially in resource limited
settings. Use of serology for the diagnosis of tuberculosis, commonly used in India, is another factor. In the present study a
commercially available serodiagnostic assay was assessed for its diagnostic value in combination with smear, culture and
clinical manifestations.
Methodology/Principal Findings: A total of 2300 subjects were recruited for the study, but 1041 subjects were excluded for
various reasons. Thus 1259 subjects were included in the study of which 470 were pulmonary tuberculosis cases (440 of 470
were culture-positive) and 789 were their asymptomatic contacts. A house-to-house survey method was used. Blood
samples were tested for IgM, IgA, and IgG antibodies using the Pathozyme Myco M (IgM), Myco A (IgA) and Myco G (IgG)
enzyme immunoassay (EIA). Out of 470 PTB cases, BCG scar was positive in 82.34%. The Mantoux test and smear positivity
rates in PTB cases were 94.3% (430/456), and 65.32% (307/470), respectively. Among the asymptomatic contacts, BCG scar
was positive in 95.3% and Mantoux test was positive in 80.66% (442/548) contacts. No contact was found falsely smear
positive. The sensitivity of IgM, IgA, and IgG EIA tests was 48.7%, 25.7% and 24.4%, respectively, while the specificity was
71.5%, 80.5%, 76.6%, respectively. Performance of EIAs was not affected by the previous BCG vaccination. However, prior
BCG vaccination was statistically significantly (p=0.005) associated with Mantoux test positivity in PTB cases but not in
contacts (p=0.127). The agreement between serology and Mantoux test was not significant.
Conclusion: The commercial serological test evaluated showed poor sensitivity and specificity and suggests no utility for
detection of pulmonary tuberculosis.
Citation: Singh S, Singh J, Kumar S, Gopinath K, Balooni V, et al. (2012) Poor Performance of Serological Tests in the Diagnosis of Pulmonary Tuberculosis:
Evidence from a Contact Tracing Field Study. PLoS ONE 7(7): e40213. doi:10.1371/journal.pone.0040213
Editor: Madhukar Pai, McGill University, Canada
Received February 24, 2012; Accepted June 2, 2012; Published July 10, 2012
Copyright: ? 2012 Singh 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: The study was supported by Indian Council of Medical Research, New Delhi (ICMR Sanction Order Number: 5/8/5/4/2005-ECD-I). 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: sarman_singh@yahoo.com
. These authors contributed equally to this work.
Introduction
Ever since WHO recognized tuberculosis (TB) a ‘global
emergency’ in 1993, implementation and expansion of WHO
supervised standardized approach to TB diagnosis and treatment
allowed more than 46 million people get cured between 1995 and
2010, averting up to 7 million deaths worldwide. Sixteenth annual
WHO global TB report showed a decrease in TB incidence, and
yet 8.8 million cases 1.4 million deaths occurred globally in 2010.
Thus, TB still remains a major global public health threat [1].
HIV-TB co-infection, multidrug-resistant (MDR) TB and emer-
gence of even more severe extensively drug resistant (XDR) TB
are further complicating the management of TB [1,2]. India had
an estimated 2.3 million (26% of global burden) TB cases in 2010,
and ranked 16thin terms of incidence rate amongst 22 highest TB
burden countries [1].
The ongoing TB epidemic reflects improper, delayed or missed
diagnosis; especially in resource limited countries. Delayed
diagnosis of TB not only postpones the required anti-tubercular
treatment (ATT), leading to more severe illness and causing
irreversible damage to affected organ(s), but also enables un-
interrupted transmission of Mycobacterium tuberculosis for longer
duration [3]. Despite impressive advances in the field of TB
diagnostics in last two decades [4], the poorly sensitive light
microscopy and poorly specific chest radiography still remain
primary means for diagnosing TB, in most of the developing
countries, including India [5]. The most signficant advances in last
few years have been liquid culture systems, and nucleic acid
amplification tests such as line probe assay and Gene-Xpert [4,6],
but high cost or sophisticated infrastructure requirements have
remained major barriers for their large scale implementation for
routine use [7].
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To overcome these limitations in current TB diagnostics,
immunological tests were initially proposed and perceived as best
point-of-care tests with potential to replace microscopy as primary
mean of rapid diagnosis of TB. Undoubtedly, if developed
successfully, serological tests have immense potential to signifi-
cantly speed up the diagnosis of TB [8]. Enzyme immune assays
(EIA) in various formats such as microwell enzyme-linked
immunosorbent assay (ELISA) and immunochromatographic tests
(ICT) have made significant impact in the early and accurate
diagnosis of several infectious diseases including HIV, malaria, and
hepatitis viral infections [9].
Since first introduction of EIA in 1976 for the diagnosis of TB,
several antigens have been tried to develop an ideal EIA [8,10–
12]. First generation EIA tests were based on crude antigens,
hence these tests exhibited low specificity. Later, an increased
understanding of genomics and proteomics led to the discovery of
new M. tuberculosis specific purified antigens having highly
immunodominant epitopes. These antigens when used singly or
in various combinations were reported to provide improved
sensitivity and specificity. But on cross validation and field
application these tests showed inconsistent results [7,13–15].
Inaccurate results were attributed to physiological stage of TB
infection [16], previous BCG vaccination, TB endemicity in the
region, exposure to other non-tuberculous mycobacteria (NTM)
[14] and host genetics or ethnicity [10].
Although, no international body has ever recommended use of
these serological tests for the diagnosis of pulmonary TB, yet more
than 70 EIA kits are available commercially for the diagnosis of
TB in high burden countries, including India [16,17]. Contradic-
tory reports in support and against the use of these tests are being
published by various authors. A meta-analysis of 67 published
studies commissioned by WHO revealed that commercial ELISA
tests exhibited highly variable sensitivity (0% to 100%) and
specificity (31% to 100%) [18]. However, no major systemic study
has been carried out from India to evaluate the sensitivity and
specificity of commercial serological tests. It is important to
understand that India is a high TB burden country and more than
half of the Indian population is exposed to the infection.
Therefore, a prospective study was planned in 2006, well before
the negative recommendation was issued by WHO against the use
of existing commercial serological kits for the diagnosis of
tuberculosis [18].
In the present study, a cohort of 2300 subjects from south Delhi,
India, was enrolled, of which 1259 subjects could be included in
the analysis. These 1259 subjects comprised of confirmed PTB
patients (470) and their family contacts (789). The sera from these
subjects were tested for IgA, IgM and IgG antibodies against a
38 kDa antigen of M. tuberculosis using pathozymeH Myco IgG, IgA
and IgM, EIA kits manufactured by Omega Diagnostic Limited,
Scotland, UK.
Results
Subjects and Clinical Parameters
A total of 2300 subjects were recruited in the study. Of these
1041 subjects had to be excluded for various reasons (Figure 1).
Hence 1259 subjects were finally included in the study. Out of
these 470 were bacteriologically confirmed PTB cases, hereafter
referred as index cases and 789 were their asymptomatic
household contacts, called contacts hereafter. Of the 470 index
cases 272 (57.9%) were males and 198 (42.1%) were females while
among the contacts 413 (52.3%) were males and 376 (47.6%)
females. All subjects were examined for BCG vaccination. Among
789 asymptomatic household contacts, 752 (95.3%) had BCG
vaccination with discernible scar while BCG scar was positive in
387 of the 470 (82.3%) patients with acute pulmonary tuberculosis,
the index cases. Mantoux/TST could be performed on 456 PTB
patients and 667 asymptomatic household contacts only. Of which
430 (94.3%) PTB cases and 476 (71.4%) contacts exhibited
Mantoux positive result (Table 1, Figure 1). Mantoux test could
not be done for remaining 14 (2.9%) PTB patients and 122
(15.5%) contacts due to their unwillingness for the test.
Mycobacteriological Findings
All patients were recruited from designated microscopy centers
(DMC) & DOTS centers of South Delhi. These DMCs undertake
microscopy of the sputum and DOTS centers provide directly
observed treatment- short course (DOTS) to all smear positive
patients, under national TB control programme in India. Thus all
our patients were smear positive at the time of registering at the
DOTS centers. We tried to recruit all the smear positive patients
in our study, as early as possible but within 15 days. All recruited
patients and their consenting asymptomatic contact were asked to
provide fresh (1 morning and 1 spot) sputum/saliva sample which
were examined in our central laboratory which is an accredited
laboratory. As shown in the flowchart only 428 smear positive
patient provided repeat sputum sample and of these 307 (71.7%)
were smear positive in our laboratory also. Additional 42 out of
831(5%) contacts were found MGIT culture positive and 4 of these
were also sputum smear positive in our laboratory during the
contact tracing. These contacts were called as co-prevalent TB
cases (see Figure 1). Out of 428 index cases, 398 (93%) were
BACTECTMMGIT 960 culture positive. Hence, a total of 440
out of 470 (93.6%) active PTB patients were culture positive.
Remaining 30 cases were bacteriologically negative in our
laboratory, but 26 of these had evidence of active PTB on Chest
X-ray, and 4 were cases of relapsed PTB, beside being smear
positive at respective DMCs. As expected, even though good
quality of sputum could not be produced by contacts, none of the
smear negative contact was culture positive, indicating high
specificity of smear microscopy.
Performance of IgM, IgA and IgG Serology
All 1259 subjects were tested for antimycobacterial antibodies as
mentioned in materials and methods section. The sensitivity,
specificity, positive predictive values (PPV), negative predictive
values (NPV) and likelihood ratio of positive (LRP) tests of 3
ELISA tests are shown in table 1 & Figure 2A. When we analyzed
individual performance of IgM, IgA and IgG among 470 PTB
cases, their sensitivity rates were 48.7%, 25.7%, and 24.4%
respectively, with respective specificities of 71.5%, 80.5% and
76.6%. When various combinations of 2 or more ELISAs were
considered for their utility in the diagnosis of PTB, the specificity
increased to 93.4% but the sensitivity was reduced to only 10.6%
(Figure 2A & 2B). Among 789 asymptomatic household contacts of
PTB patients, IgM, IgA and IgG EIAs were positive in 28.5%,
19.5% and 23.4% respectively (Table 1, Figure 2B), showing very
low specificity in this cohort of asymptomatic family contacts.
Positive predictive values (PPV) for IgM, IgA and IgG were
50.4%, 44% and 38.3% respectively, while the negative predictive
values (NPV) were 70%, 64.5% and 64.9% respectively (Table 1).
Likelihood ratio of positive (LRP) test helps to predict the
likelihood of true positive result allowing better interpretation of
the test results. Likelihood ratio for positive test for IgM, IgA and
IgG were 1.7, 1.3 and 1.0 respectively (Table 1). Low PPV, NPV
and LRP values in all three EIA tests further revealed that
diagnostic potential of these serological tests is very low.
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Serology vs Mantoux Test
As all three EIA kits measured anti-mycobacterial humoral
(antibody) immune response in the serum, their performances
were also compared with Mantoux test which measures cellular
immune response against exposure to M. tuberculosis. The
Mantoux test was found to be more sensitive tool then serology
with 94.3% sensitivity but as expected its specificity was low
(28.6%) when 10 mm induration size was taken as cut-off.
However, its specificity improved to 70.6% when induration
diameter of $15 mm was taken as cut-off size. Even at this cut-
off its sensitivity remained 72.5% which was better than any
single serological test (Table 1). Mantoux test showed much
better PPV (62.7%), NPV (78.9%) and LRP (2.5) test values as
compared to serology. Statistical inter-test agreement was also
determined using percentage agreement and Cohen’s Kappa
coefficient ‘k’ (Table 2). Out of 456 PTB patients who were
subjected to Mantoux evaluation, 94.3% patients demonstrated
positive result; and out of 667 household contacts 71.4%
showed positive Mantoux results (Table 3). On agreement
assessment with Mantoux test results, IgM, IgA and IgG EIA
showed only 48% (k=20.035), 27% (k=20.026) and 28%
(k=20.023) agreement respectively in PTB cases. The negative
kappa coefficient values signify that any agreement between
results of any two serological tests is equal or worse than a
chance finding. Among asymptomatic household contacts also,
all Cohen’s Kappa (k) values were just above ‘0’ showing a very
poor agreement between any two tests.
Table 1. Performance of IgM, IgA, and IgG ELISA and Mantoux test in bacteriologically confirmed TB patients and Contacts.
ELISA (n=470)
PTB
(n=470)
Contacts
(n=789)
Sensitivity %
(95% CI)
Specificity %
(95%CI)
PPV#(%)
(95% CI)
NPV#(%)
(95% CI)LRP#(95% CI)
(Pos)229 (48.7) 225 (28.5) 48.7 (44.2–53.2)71.5 (68.2–74.5) 50.4 (45.8–55)70.0 (66.1–73.1)1.7 (1.6–1.7)
IgM
(Neg)241 (51.2) 564 (71.4)
(Pos) 121 (25.7) 154 (19.5)25.7 (22–29.8)80.5 (77.5–83.1)44.0 (38.2–49.9)64.5 (61.4–67.4)1.3 (1.2–1.4)
IgA
(Neg) 349 (74.2)635 (80.4)
(Pos) 115 (24.4)185 (23.4)24.4 (20.8–28.5)76.6 (73.4–79.3) 38.3 (33–43.9)62.9 (59.8–65.9)1.0 (0.97–0.99)
IgG
(Neg) 355 (75.5)604 (76.5)
(Pos) 87 (18.5) 79 (10.0)18.5 (15.2–22.2) 89.9 (87.7–91.8) 42.4 (44.8–49.8)64.9 (62.0–67.7)1.8 (1.6–2.0)
IgM &IgA
(Neg)383 (81.4)710 (89.9)
(Pos) 71 (15.1)88 (11.1) 15.1 (12.1–18.6)88.8 (86.4–90.8) 44.6 (37.1–52.4)63.7 (60.8–56.5)1.3 (1.1–1.)
IgM &IgG
(Neg)399 (84.8)701 (88.8)
(Pos)62 (13.9)83 (10.5) 13.1 (10.4–16.5)89.4 (87.1–91.4) 42.7 (35–50.9)63.3 (60.5–66.1)1.2 (0.99–1.5)
IgA &IgG
(Neg)408 (86.1)706 (89.4)
(Pos)50 (10.6) 52 (6.5) 10.6 (8.1–13.7)93.4 (91.4–94.9) 49.0 (39.5–58.5)63.7 (60.8–66.4)1.6 (1.1–2.3)
IgM, IgA &IgG
(Neg)420 (89.3)737 (93.4)
(Pos)
Any EIA+
295 (62.8) 366 (46.4)62.8 (58.3–67.0)53.6 (50.1–57.1)44.6 (40.9–48.4) 70.7 (66.9–74.2)1.35 (1.34–1.37)
(Neg)175 (37.2) 423 (53.6)
(Pos) 430 (94.3)476 (71.36)94.3 (91.8–96.0)28.6 (25.3–32.2) 47.5 (44.2–50.7)88.0 (83–91.7)1.3 (1.31–1.32)
Mantoux*1
(Neg)26 (5.7)191 (28.6)
(Pos)330 (72.4)196 (29.4) 72.5 (68.1–76.3)70.6 (67.1–74.0)62.7 (58.5–66.8)78.9 (75.4–82.0)2.5 (2.4–2.5)
Mantoux*2
(Neg)126 (27.6) 471 (70.6)
(Pos): positive; (Neg): negative; CI: confidence interval;
#PPV: positive predictive value;
##NPV= Negative predictive value; LRP: likelihood ratio for positive test;
*Mantoux test was done only in 456 out of 470 PTB patients and 667 out of 789 asymptomatic household contacts; Mantoux*1when Mantoux results with skin reaction
indurations size $10 mm were interpreted as positive result, whereas in Mantoux*2results with reaction indurations size $15 mm were considered as Mantoux
positive.
+Any EIA: means subjects detected positive by at least one of three (IgG/IgM/IgA) EIA tests. Values in parenthesis are percentage with 95% CI values.
doi:10.1371/journal.pone.0040213.t001
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Serology, Mantoux Test and Prior BCG Vaccination
In India BCG vaccination is given at birth under the expanded
programme of immunization to all. BCG scar was positive in
82.3% (387/470) PTB cases and 95.3% (752/789) asymptomatic
household contacts. Effect of BCG was also observed on the
performance of Mantoux test. Statistically significant association
between BCG and Mantoux test was observed in PTB cases
(p=0.005), when results were interpreted taking 10 mm cut-off
induration size (Table 3). But no statistically significant association
was observed between BCG vaccination and results of serology
[IgM (p=0.8923), IgA (p=0.0665) and IgG EIA (p=0.9308)].
However, IgM plus IgA combination showed statistically signifi-
cant (p=0.0387) difference between BCG scar positive and scar
negative PTB patients, indicating that scar negative persons were
more likely to develop PTB and that they were more likely to be
Figure 1. The flow chart showing the number of subjects recruited and finally enrolled in the study with details of co-prevalent TB
in asymptomatic family contacts of the index patients, rate of BCG vaccination and Mantoux test findings.
doi:10.1371/journal.pone.0040213.g001
Figure 2. Seropositivity rates for IgA, IgM and IgG antibodies individually and in various combinations in confirmed pulmonary
Tuberculosis patients (n=470) [panel A] and in asymptomatic family contacts (n=789) [panel B].
doi:10.1371/journal.pone.0040213.g002
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IgM & IgA seropositive. Similar, association was observed in
asymptomatic contacts also (p=0.0186) (Table 3).
Discussion
Recognition of diagnostic potential of serological tests for TB
has long history which dates back to 1898, when Arloing
successfully agglutinated antibodies from TB patients’ sera [19].
After the slow progress for several decades, this concept got
significant boost with the introduction of enzyme-linked immuno-
sorbent assay (ELISA) test for TB by Nassau et al in 1976 [12].
Since then several mycobacterial immunodominant antigens have
been identified and evaluated in different ways improving on from
increased understanding of anti-mycobacterial humoral immune
response against M. tuberculosis. Many diagnostic assays based on
single and multiple M. tuberculosis specific purified antigens have
been evaluated but with variable specificity and sensitivity [13,20–
22]. Use of purified and recombinant antigen(s) of various
infectious agent has improved the sensitivity and specificity but
such specific antigens are yet to be discovered for TB diagnosis
[10,23]. Despite low sensitivity and specificity of serology for the
diagnosis of tuberculosis, India remains a major user of serology,
specially the private sector. It is mainly because poor regulation of
diagnostics, requirement of less skilled technicians, minimum
biohazard and marketing by the commercial organizations [17].
The aim of our study was to evaluate performance of three
serological tests on confirmed active pulmonary tuberculosis (PTB)
casesandtheirasymptomatichouseholdcontacts.Significantlyhigh
sample size of, active PTB patients (470) and their asymptomatic
household contacts (789), was major strength of this study.
ThesensitivityandspecificityofPathozymeMycoM(IgM),Myco
A (IgA) and Myco G (IgG) enzyme immunoassay (EIA) have been
shown to be highly variable in previous studies in different settings,
albeit on smaller sample size [24–27]. Pottumarthy et al in New
Zealand reported sensitivity of 18%, 41% and 55% respectively for
Pathozyme Myco M, Myco A and Myco G. Using a very small
sample size of 44 PTB patients, they calculated specificity of 100%,
72% and 90% [24]. The same kits were also evaluated on 94 PTB
cases in Pakistan by Butt et al, showing sensitivity of 67% and 46%
andspecificityof98%and93%forMycoMandMycoG[25].Imaz
etalfromArgentinaalsoevaluatedPathozymeMycoM,MycoAand
Myco G EIA on only 58 PTB patients in a hospital setting and
demonstrated high specificity of 93.3%, 97.8% and 100% respec-
tively. Their respective sensitivity rates were 29.4%, 76.5% and
82.3% for 17 smear positive PTB cases, and 31.7%, 34.2% and
48.8% for 41 smear negative PTB cases [26]. No study was carried
out at a community level in a specific cohort of patients. Results of
Table 2. Agreement assessment of EIAs vis-a `-vis Mantoux test.
ELISA Results
Pulmonary Tuberculosis PatientsAsymptomatic Household Contacts
Mantoux (n=456)#
Mantoux (n=667)#
Total Pos, n=430 Neg, n=26 PA* Cohen’s kappaTotal Pos, n=476 Neg, n=191) PA*Cohen’s kappa
(Pos) 228 (50.0) 211 (49.1)17 (65.4)48.2 20.035 (20.078–0.007) 195 (29.2) 141 (29.6) 54 (28.3) 41.7 0.009 (20.043–0.062)
IgM
(Neg) 228 (50.0) 219 (50.9)9 (34.6)472 (70.8) 335 (70.4)137 (71.7)
(Pos) 120 (26.3) 109 (25.3)11 (42.3)27.2 20.026 (20.052–0.00178) 134 (20.1) 103 (21.6)31 (16.2)39.4 0.035 (20.008–0.079)
IgA
(Neg) 336 (73.7) 321 (74.7)15 (57.7) 533 (79.9) 373 (78.4) 160 (83.8)
(Pos)115 (25.2) 109 (25.3)6 (23.1) 28.3 0.003 (0.022–0.029) 164 (24.6) 120 (25.2)44 (23.0)40.00.015 (20.034–0.063)
IgG
(Neg)341 (74. 8) 321 (74.7)20 (76.9)503 (75.4) 356 (74.8)147 (77.0)
(Pos)87 (19.1)78 (18.1) 9 (34.6)20.8 20.023 (20.044–0.001)71 (10.6) 57 (12.0)14 (7.3)35.1 0.028 (20.003–0.060)
IgM &IgA
(Neg)369 (80.9) 352 (81.9)17 (65.4) 596 (89.4) 419 (88.0) 177 (92.7)
(Pos) 71 (15.6)65 (15.1) 6 (23.1) 18.6 20.011 (0.03–0.009)80 (12.0) 59 (12.4)21 (11.0) 34.30.009 (20.025–0.042)
IgM &IgG
(Neg)385 (84.4) 365 (84.9) 20 (76.9)587 (88.0) 417 (87.6) 170 (89.0)
(Pos)62 (13.6)56 (13.0) 6 (23.1) 16.7 0.013 (20.031–0.005)75 (11.2)62 (13.0)13 (6.8)36.00.038 (0.006–0.071)
IgA &IgG
(Neg)394 (86.4) 374 (87.0) 20 (76.9)592 (88.8) 414 (87.0)178 (93.2)
(Pos)50 (10.9)44 (10.2)6 (23.1)14.0 20.016 (–0.032–0.001)48 (7.2)39 (8.2) 9 (4.7)33.1 0.021 (20.005–0.047)
IgM, IgA& IgG
(Neg) 406 (89.1) 386 (89.8) 20 (76.9) 619 (92.8) 437 (91.8)182 (95.3)
Pos 293 (64.3) 274 (63.7)19 (73.1)61.6 20.027 (20.082–0.028) 315 (47.2) 225 (47.3)90 (47.1) 48.90.001 (20.066–0.068)
Any EIA**
Neg163 (35.7) 156 (36.3)7 (26.9)352 (52.8) 251 (52.7)101 (52.9)
*PA: means percentage agreement; Any EIA pos means PTB cases and asymptomatic household contacts if positive by at least one of three EIA (IgM, IgA & IgG);
#Mantoux test was done only in 456 out of 470 PTB patients and 667 out of 789 asymptomatic household contacts. Values in in parenthesis are percentage.
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such small studies have been exploited extensively by the commer-
cial firms in most of the TB endemic countries for their own benefit.
However, some studies, even from TB non-endemic countries also
showed very poor sensitivity but disregarded by commercial firms
[27]. Recently we reviewed the situation of TB serology market in
Asia and the searchresults revealed thatmorethan 73brands ofTB
serology kits are being marketed either in microwell ELISA or
immunochromatographic (ICT) test formats [17].
Our study clearly shows that serology has no place in the
diagnostic algorithm of pulmonary tuberculosis. In confirmed PTB
patients, Pathozyme Myco A (IgA), and Myco G (IgG) EIAs
demonstrated barely 25.7% and 24.4% sensitivities respectively,
with 80.5% and 76.6% specificities. The Myco M (IgM) EIA
showed slightly better (48.7%) positivity in PTB cases, but at the
same time its specificity was also very low (71.4%) (Table 1). The
slightly higher positivity could also be due to well known
interfering antibodies like rheumatoid factor. Though we did not
include disease controls such as patients with autoimmune
diseases; and if used, the specificity could have gone further down.
The sensitivity and specificity rates of all three Pathozyme Myco
EIAs shown in our study were generally lower than those reported
by other investigators [24–27]. This difference could be explained
on the basis of inclusion of asymptomatic household contacts of
PTB cases, which were living with PTB patients in same
households and so were more likely to be exposed to M. tuberculosis.
Moreover, as mentioned above these published studies used very
small sample size and PTB patients were compared with non-TB
patients. However, it is important to highlight that even after using
combination of IgG/IgM/IgA, 37.3% of confirmed PTB patients
could not be detected by any of the EIAs and showed false
negative results (Table 1). This has serious implications for any TB
control programme, i.e more than one third infectious PTB
patients could be missed, if the serology is used as the sole criteria
for administering anti-tubercular treatment. Our study clearly
showed that even the sensitivity of light microscopy was better
Table 3. Effect of BCG vaccination on TB EIAs & Mantoux test.
Pulmonary Tuberculosis PatientsAsymptomatic Household Contacts
ELISA Results
History of BCG (n=470)History of BCG (n=789)
Total
Scar Pos,
(n=387)
Scar Neg,
(n=83)
p* valueTotal
Scar Pos,
(n=752)
Scar Neg,
(n=37)p* value
(Pos)229 (48.7) 188 (48.6)41 (49.4) 0.8923 225 (28.5)216 (28.7)9 (24.3) 0.5629
IgM
(Neg) 241 (51.3)199 (51.4)42 (50.6) 564 (71.5)536 (71.3)28 (75.7)
(Pos) 121 (25.7)93 (24.0)28 (33.7)0.0665 154 (19.5)150 (19.9)4 (10.8) 0.1712
IgA
(Neg) 349 (74.3)294 (76.0)55 (66.3) 635 (80.5) 602 (80.1)33 (89.2)
(Pos) 115 (24.5) 95 (24.5)20 (24.1)0.9308 185 (23.5)180 (23.9) 5 (13.5)0.1441
IgG
(Neg) 355 (75.5) 292 (75.5)63 (75.9) 604 (76.5)572 (76.1)32 (86.5)
(Pos) 87 (18.5)65 (16.8) 22 (26.5)0.038779 (10.0)79 (10.5)00.0186
IgM &IgA
(Neg)383 (81.5) 322 (83.2)61 (73.5) 710 (90.0)673 (89.5)37 (100)
(Pos) 71 (15.1)57 (14.7) 14 (16.9)0.621588 (11.2)87 (11.6) 1 (2.7)0.0777
IgM &IgG
(Neg)399 (84.9) 330 (85.3)69 (83.1)701 (88.8)665(88.4)36 (97.3)
(Pos)62 (13.2)49 (12.7)13 (15.7) 0.4635 83 (10.5)82(10.9)1 (2.7)0.0972
IgA &IgG
(Neg) 408 (86.8)338 (87.3) 70 (84.3)706 (89.5) 670 (89.1) 36 (97.3)
(Pos) 50 (10.6) 38(9.8)12 (14.5)0.2140 52 (6.6)52 (6.9)00.0766
IgM, IgA& IgG
(Neg)420 (86.4)349(90.2)71 (85.5)737 (93.4)700 (93.1) 37 (100)
Pos 295 (62.8)243 (62.8)52 (62.7)0.9809 366 (46.4)350 (46.5)16 (43.2)0.6944
Any EIA**
Neg175 (37.2)144 (37.2) 31 (37.3)423 (53.6)402 (53.6)21 (56.8)
Pos 429 (94.1)358 (95.7)72 (87.8) 0.005 476 (71.4) 454 (70.8)22 (84.6) 0.1274
Mantoux#
Neg27 (5.9)16 (4.3)10(12.2) 191 (28.6)187 (29.2)4 (15.4)
*P value indicated two tail p value calculated from Pearson chi-square test and Fisher test;
**Any EIA pos denotes at least one of three EIA (IgM, IgA & IgG) tests was positive;
#Mantoux test was done only in 456 out of 470 PTB patients and 667 out of 789 asymptomatic household contacts. Values in parenthesis are percentage.
doi:10.1371/journal.pone.0040213.t003
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(71.7%) than serology which could detect mycobacteria in the
sputum of PTB cases as against 62.5% by serology. Dowdy et al
[28] also concluded that smear microscopy still remains the most
cost-effective initial diagnostic test for PTB similar to our findings.
We did not find any false positive smear result in this study.
Diagnostic potential of a test in clinical practice also depends on
its predictive values, and likelihood ratio of positive test. High
positive predictive values of a test make the test useful in
strengthening the clinical suspicion of disease, while high negative
predictive values of test makes the test useful in exclusion of disease
in negative cases [24]. The most commonly used IgM kit
demonstrated positive and negative predictive values of just
50.4% and 70.0% respectively. Indicating that this test was of
no help in confirming TB infection, and it failed to correctly rule
out the TB in 30% asymptomatic contacts. Predictive values of
other two EIAs were rather less confirmatory (Table 1). Likelihood
ratio of positive (LRP) test is also an important statistical method to
better evaluate the diagnostic test [27]. In our study, LRP values
for serological tests were less accurate (ranging from 1–1.8 only for
various serology combinations) than the Mantoux test which alone
has a LRP of 2.5.
The Mantoux test is century old and is an inexpensive test for
detecting the latent TB infection. This test showed sensitivity of
94.3% and specificity in asymptomatic contacts as 28.6%, which
may be explained by use of crude antigen and exposure to
mycobacteria both from environment and from the index patients
residing in the same household. Previous BCG vaccination also
seemingly had positive impact on Mantoux reaction, as the
positivity rate in scar positive and scar negative patients was
significantly (p=0.005) different. When the induration size of
$15 mm diameter was taken as cut-off value, the specificity of the
test improved significantly to 70.6%, maintaining a sensitivity of
72.5% (Table 1). Similar observations are reported by Wang et al
[29].
Many commercial serological TB tests are available on the
market, based on small, in-house studies. Poor regulation allows
the widespread use of these tests [17]. WHO in its reports
mentioned that ‘‘a vast majority of studies were either sponsored
by industry, involved test manufacturers, or failed to provide
information on industry sponsorship’’ [18]. Although, no country
ever recommended their use, several serological tests for TB
diagnosis are marketed and widely used in many parts of the world
[9,30], especially in developing countries like India with weak
regulatory systems [30,31]. However, after the advisory of WHO,
the Government of India has taken some concrete steps for
banning these serological tests. Nevertheless, it remains to be seen
if the ban will be successfully implemented and enforced.
Conclusion
The evidence provided in this study suggests that, none of the
antibody tests, alone or in combination, perform well enough to
replace sputum smear microscopy. These tests thus have little or
no role to play in the diagnosis of pulmonary TB. Our study
findings support the recent negative policy recommendations
against TB serological tests by WHO.
Materials and Methods
Ethics Statement
Ethical committee of the All India Institute of Medical Sciences
(AIIMS), New Delhi approved the study protocol in accordance
with National Guidelines by Indian Council of Medical Research.
All the subjects were recruited with their signed consent on
ethically approved consent form informed in both Hindi and
English after explaining the purpose and implications of the study
by the well trained field investigator.
Study Design and Subject Recruitment
The study was conducted between 2006 and 2010 at the TB
Laboratory, Clinical Microbiology Division, Department of
Laboratory Medicine, All India Institute of Medical Sciences,
New Delhi in collaboration with designated microscopy centers
(DMC) and DOTS centers of South Delhi region (Khanpur,
Dakshinpuri, Madangir, Safdarjung, and Shahpur Jat). After
approval of the study from the central TB control division of
Government of India, we approached the DMCs of the respective
area to identify the smear positive patients diagnosed at their
respective DMC within last two weeks. All the sputum smear
positive patients were contacted at their place of residence, their
detailed clinical history was noted and after written consent, 508
index PTB cases and 1792 family contacts were recruited.
However, after further work-up 62 PTB patient refused consent
for inclusion in the study, and 18 had no regular house hold and
thus these were excluded. Similarly out of 1792 recruited family
contacts, 961 refused to give blood sample and 42 were found to
have co-prevalent TB and thus grouped into the PTB group
(please see Figure 1). Finally a total of 1259 subjects were enrolled
in the study. All PTB cases, whether untreated, relapse, or under
treatment (but not responding to treatment) were included in the
study. All the demographic details and relevant clinical symptoms,
signs and duration were documented in predesigned subject
information form.
Case Definition
TB patients were defined as PTB cases where infection of lungs,
pleural cavities or respiratory tracts with M. tuberculosis occurs and
the disease is diagnosable with chest X-ray, smear microscopy,
culture or had favourable response to antitubercular treatment.
Household contacts in this study were defined as all the family
members/tenants/groups generally living together in the same
shelter with same front door and who live in prolonged/intense
contact with the PTB patient [32]. Among the household contacts
only contacts who had no symptoms of TB infection in
preliminary investigation were included in the present study.
Sample Collection and Processing
Preliminary diagnosis of PTB was made at local designated
microscopy centers (DMC) after examining patient’s morning or
spot sputum samples. After obtaining information of smear
positive patients we noted the contact details of the patients and
field worker contacted the patient’s family and fixed the
appointment for sampling. A repeat sputum (1 morning plus 1
spot) sample and 5 ml blood were collected (before doing Mantoux
test) in sterile containers and samples transported on the same day
to TB Laboratory, Clinical Microbiology Division at AIIMS for
further processing. The asymptomatic healthy contacts who could
not produce good quality sputum, even the saliva samples were
accepted for the study. The sputum/saliva samples were processed
after decontamination by modified Petroff’s (NALC/NaOH)
method [33]. The processed sputum samples were inoculated in
MGIT (Mycobacterium Growth Indicator Tube) of automated
BACTECTMMGIT 960 culture system following manufacturer’s
instructions (Becton Dickinson, USA). Ziehl-Neelson (ZN) staining
followed by microscopy was done on both direct and decontam-
inated sputum samples for acid fast bacilli (AFB). Serum was
separated from the blood samples by centrifugation and stored at
220uC for further use in ELISA avoiding repeated freezing and
thawing.
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Tuberculin Skin Test
Tuberculin skin test (TST) or Mantoux test was carried out by
intradermally injecting 0.1 ml of 5TU (Span Diagnostics Ltd,
India) purified protein derivative (PPD) into the volar surface of
the forearm. While injecting PPD it was ensured that level of
tuberculin syringe needle was facing upward so that a pale
elevation of the skin (a wheal), 6 to 10 mm in diameter, was
formed. Mantoux test was done only after withdrawing blood
sample. The patients were instructed not to apply any soap/
detergent or wash the area to avoid itching and scratching for the
next 48 hours. The injection site was encircled by permanent
marker and reaction induration (palpable, raised, hardened area
or swelling) was measured in millimeter (mm) after 48–72 hours
[32,34]. The test was performed by well trained field investigators.
Recording the Details of BCG Vaccination
BCG status was determined using visual inspection of scars. The
subjects with clearly visible scar were considered as BCG
vaccinated and remaining without scar as non-vaccinated.
TB-ELISA
We screened published literature for performance of dozens of
commercial serological tests offered for sale in Indian market and
selected PathozymeH Myco IgG, IgA and IgM, EIA kits
manufactured by Omega Diagnostic Limited, Scotland, UK
[35]. These kits were selected because of more/or widespread
use, comparative better performance as available on the public
domain and combination of antigens used in it [24]. These kits are
based on two highly purified immunodominant antigens, the cell
wall lipoarabinomannan (LAM) antigen which, and a 38-kDa
mycobacterial recombinant antigen [35]. The kits claimed to be
having 91% specificity and 72% sensitivity [36]. The EIA tests
were performed according to the instructions provided in kits’
manual (Omega diagnostics limited, Scotland, UK). All three EIA
kits were evaluated simultaneously with the same serum samples
aliquots stored at 220uC.
Statistical Analysis
For proper analysis of performance, ELISA tests were evaluated
first on asymptomatic household contacts and then on confirmed
PTB cases. Sensitivity and specificity was determined using
confirmed PTB cases and asymptomatic contacts as positive and
negatives references. Other statistical analysis, such as Positive
predictive values (PPV), negative predictive value (NPV) and
likelihood ratio for positive (LRP) test were also calculated with
95% CI (confidence intervals). PPV (also called precision rate) is
the proportion of subjects with positive test results who are
correctly diagnosed for infection. NPV is defined as the proportion
of subjects with negative test result who are correctly ruled out of
infection. Higher PPV and NPV denote more correct assessment.
Likelihood ratio of positive (LRP) test helps to predict the
likelihood of true positive result allowing the clinician to better
interpret the results of the diagnostic test. A LRP of greater than 1
indicates the test result is associated with the presence of disease
and less than 1 means the test result is associated with the absence
of the disease. Percentage agreement was assessed between the
results of Mantoux test and EIAs. To rule out the proportion of
agreement by chance, Cohen’s kappa test was used. To check the
effect of BCG vaccination on the performance of EIAs and
Mantoux test, Pearson Chi-square test and exact mid-p test were
used. P-value ,0.05 was considered statistically significant.
STATA SE.9 software was used for all statistical analysis.
Acknowledgments
Authors would like to thank Mr. Hem Kumar, Satendra Saini and
Virender Kapil for technical assistance, Mr. Deepak Kumar, Nand
Kishore and Shakir Ahmad for field work, medical officers from
Safdarjung Hospital, Malaviya Nagar Colony Hospital and Charak Palika
Hospital and technical staff of designated microscopy and DOTS centers of
Dakshinpuri, Khanpur and Madangir (all Delhi) for their support in
collecting the data and for enrollment of patients from their respective
DOTS centers for the study. Authors also wish to thank Dr. LS Chauhan,
the then Director Central TB division, Government of India and Dr. RP
Vasisht then State TB Officer, Delhi for allowing this study under their
control.
Author Contributions
Conceived and designed the experiments: SS SK KG NS. Performed the
experiments: SK JS KG VB. Analyzed the data: SK JS NS KG SS KM.
Contributed reagents/materials/analysis tools: SS. Wrote the paper: SS
SK.
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