Article

Development and evaluation of a novel multiple-antigen ELISA for serodiagnosis of tuberculosis

State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, No. 220 Handan Road, 200433 Shanghai, PR China.
Tuberculosis (Edinburgh, Scotland) (Impact Factor: 2.71). 07/2009; 89(4):278-84. DOI: 10.1016/j.tube.2009.05.005
Source: PubMed

ABSTRACT

In this study, we describe the development and evaluation of a novel multiple-antigen ELISA for rapid diagnosis and screening of active tuberculosis (TB). The humoral immune responses of 136 active TB patients and 57 healthy subjects against antigens Rv3425, 38kDa and lipoarabinomannan (LAM) from Mycobacterium tuberculosis H37Rv were examined by ELISA. Three essential results were obtained. (i) Rv3425 antigen is a potential candidate for serodiagnosis of active TB. Of 136 active TB patients, Rv3425 antigen provided a sensitivity of 31.6%, lower than that of LAM antigen, but higher than that of 38kDa antigen, with an overall specificity of 100%. (ii) For 62 smear-negative pulmonary TB patients and 15 extra-pulmonary TB patients, the multiple-antigen test provided a sensitivity of 43.5% and 26.7%, respectively, representing an improvement over acid-fast bacilli (AFB) smear-based diagnosis. (iii) Compared with the single-antigen ELISA and the two available commercial kits, the multiple-antigen test offered the highest accuracy (71.0%). In conclusion, the multiple-antigen ELSIA test based on Rv3425, 38kDa, and LAM antigens is a potentially useful tool for the serodiagnosis and screening of active TB. Combinations of Rv3425 with other mycobacterial antigens may also be worthy of further investigation.

Full-text

Available from: Jun-Wei Zhao
DIAGNOSTICS
Development and evaluation of a novel multiple-antigen ELISA
for serodiagnosis of tuberculosis
Shu-Lin Zhang
a
, Jun-Wei Zhao
b
, Zhan-Qiang Sun
a
, En-Zhuo Yang
a
, Ji-Huan Yan
c
, Qi Zhao
d
,
Guo-Long Zhang
e
, Hong-Mei Zhang
a
, Yuan-Ming Qi
b
, Hong-Hai Wang
a
,
*
, Qing-Wen Sun
a
,
*
a
State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, No. 220 Handan Road, 200433 Shanghai, PR China
b
College of Life Sciences, Zhengzhou University, Zhengzhou, Henan, PR China
c
Hebei International Traveling Hygiene Health Center, Shijiazhuang, Hebei, PR China
d
Linyi Chest Hospital, Linyi, Shandong, PR China
e
Henan Chest Hospital, Zhengzhou, Henan, PR China
article info
Article history:
Received 3 March 2009
Received in revised form
28 April 20 09
Accepted 24 May 2009
Keywords:
Tuberculosis
ELISA
Rv3425
38 kDa
LAM
Serodiagnosis
summary
In this study, we describe the development and evaluation of a novel multiple-antigen ELISA for rapid
diagnosis and screening of active tuberculosis (TB). The humoral immune responses of 136 active TB
patients and 57 healthy subjects against antigens Rv3425, 38 kDa and lipoarabinomannan (LAM) from
Mycobacterium tuberculosis H37Rv were examined by ELISA. Three essential results were obtained. (i)
Rv3425 antigen is a potential candidate for serodiagnosis of active TB. Of 136 active TB patients, Rv3425
antigen provided a sensitivity of 31.6%, lower than that of LAM antigen, but higher than that of 38 kDa
antigen, with an overall specificity of 100%. (ii) For 62 smear-negative pulmonary TB patients and 15
extra-pulmonary TB patients, the multiple-antigen test provided a sensitivity of 43.5% and 26.7%,
respectively, representing an improvement over acid-fast bacilli (AFB) smear-based diagnosis. (iii)
Compared with the single-antigen ELISA and the two available commercial kits, the multiple-antigen test
offered the highest accuracy (71.0%). In conclusion, the multiple-antigen ELSIA test based on Rv3425,
38 kDa, and LAM antigens is a potentially useful tool for the serodiagnosis and screening of active TB.
Combinations of Rv3425 with other mycobacterial antigens may also be worthy of further investigation.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Tuberculosis (TB) is still one of the greatest health care problems
in the world. The development of a rapid and accurate test for the
diagnosis of TB, in particular smear-negative pulmonary and extra-
pulmonary TB, is a priority for TB control.
1
To date, however, there
is no simple, rapid, sensitive and specific test that can differentiate
active TB from latent infection and slowly progressive TB. Although
the detection of acid-fast bacilli (AFB) by a microscopic examination
of sputum smears is a simple and relatively quick means of
detecting active TB, the sensitivity is compromised because greater
than 10
4
bacilli per ml of sputum is required for reliable detection.
2
Traditional diagnostic techniques based on the isolation of the
tuberculous bacillus in culture media are time consuming, and it is
necessary to wait for several weeks to obtain a result.
3
Therefore,
the search for rapid and reliable diagnostic tests for active TB based
on the examination of sputum, blood and other clinical specimens
has still been the focus of TB control.
Several novel diagnostic techniques have been investigated in
recent years to determine their abilities to improve the diagnosis of
TB. These techniques include the detection of Mycobacterium
tuberculosis in clinical specimens by nucleic acid amplification
(NAA) tests, such as polymerase chain reaction (PCR) and other
methods for amplifying DNA and RNA, as well as immune reactions
based on the cell-mediated immune response (CMI) or humoral
immune responses. Commercial NAAs yield variable sensitivity
estimates and are expensive, making this technology of limited
benefit in developing countries.
4
In vitro tests based on CMI make
possible the early detection of individuals with latent infection
5–7
and are of great importance for contact tracing and screening of
high-risk groups in a setting of low endemicity.
8–11
However, this
method is not an ideal alternative to microscopy and culture in
*
Corresponding authors. Tel.: þ86 21 6564 3777; fax: þ86 21 6564 8376 (H.-H.
Wang). Tel.: þ86 21 55665073; fax: þ86 21 65648376 (Q.-W. Sun).
E-mail addresses: hhwang@fudan.edu.cn (H.-H. Wang), biolotus@163.com
(Q.-W. Sun).
Contents lists available at ScienceDirect
Tuberculosis
journal homepage: http://intl.elsevierhealth.com/journals/tube
1472-9792/$ see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tube.2009.05.005
Tuberculosis 89 (2009) 278–284
Page 1
developing countries, as large proportions of the populations in
such countries are likely to harbor latent infection with M. tuber-
culosis.
11–13
A serological test would be an attractive alternative diagnostic
method; such a test would be rapid, easy to perform and user
friendly, and could be implemented easily in the conditions found
in developing countries as, unlike NAA, it makes possible to use
blood, which is easier to obtain than respiratory samples or other
organic fluids and tissues, and unlike CMI-based assays, it does not
require viable cell cultures. In recent years, considerable progress
has been made in identifying promising antigens for a serodiag-
nostic test for TB.
12,1 4,15
Furthermore, based on the theory of anti-
body profiles of TB, much attention has been focused on screening
multiple antigens and multi-epitopes specific for M. tuberculosis to
develop novel serodiagnostic tools for TB.
16–19
Our previous study revealed a candidate antigen Rv3425 for the
serodiagnosis of TB, which is a member of the PPE protein family
encoded by the RD11 region of M. tuberculosis. The gene only exists
in pathogenic mycobacteria and is not found in BCG or other
mycobacteria. The result demonstrated the immunodominant
nature of recombinant Rv3425 protein, based on its reactivity with
sera obtained from patients with both pulmonary and extra-
pulmonary TB and negligible reactivity with sera obtained from
healthy controls.
15
Further study showed that Rv3425 protein
induced an increased Th1/Th2 type immune response in mice,
characterized by an elevated concentration of IFN-
g
in antigen
stimulated splenocyte culture and a strong IgG1 antibody
response.
20
These results provide evidence on the immunogenicity
of the PPE protein Rv3425. However, there have been no reports on
the potential usefulness of Rv3425 together with other immuno-
dominant antigens. In this study, we combined Rv3425 antigen
with two well-known immunodominant antigens including 38 kDa
protein and lipoarabinomannan (LAM) from M. tuberculosis H37Rv
to detect antibodies in the active TB patients and healthy subjects,
and then evaluated the efficacy of the novel multiple-antigen ELISA
for rapid diagnosis and screening of active TB.
2. Materials and methods
2.1. Serum samples and patients
A total of 193 serum samples (n ¼ 193) from HIV-seronegative
individuals were studied. Serum samples (n ¼ 136) from active TB
patients (age range, 1–86 years) were collected from Henan Chest
Hospital, PR China and Linyi Chest Hospital, Shandong, PR China.
There were 77 men and 59 women. In this study, active TB patients
were diagnosed by the isolation and identification of M. tuberculosis,
as well as by clinical and radiological findings. Mycobacterial isolates
were obtained from Lowenstein–Jensen cultures, identified to the
species level by biochemical procedures,
21
and further confirmed by
genotyping based on the 16S–23S rRNA gene internal transcribed
spacer (ITS) sequence.
22
A total of 136 active TB patients were
confirmed and classified into three groups: (i) smear-positive for
acid-fast bacilli (AFB) and culture-positive pulmonary TB (n ¼ 59);
(ii) smear-negative culture-positive pulmonary TB (n ¼ 62); (iii)
extra-pulmonary TB (n ¼ 15). The last group was again divided into
subgroups: scrofula (n ¼ 4), pleural TB (n ¼ 3), tuberculous menin-
gitis (n ¼ 3), bone TB (n ¼ 2), intestinal TB (n ¼ 2), and cutaneous TB
(n ¼ 1). All of the patients had not yet been started with antituber-
culous chemotherapy when the serum samples were taken.
Serum samples (n ¼ 57) of healthy control subjects (age range,
16–72 years) were obtained from Hebei International Traveling
Hygiene Health Center, PR China. There were 35 men and
22 women. All of the control subjects had not previously suffered
from TB, and had negative chest X-rays and negative sputum
culture results for M. tuberculosis. Thirty-nine of these had previ-
ously been vaccinated with Mycobacterium bovis BCG, 39 were PPD
positive, and 18 were PPD negative at the time the serum samples
were taken. Sera were stored at 20
C.
2.2. Cloning, expression and purification of the recombinant
protein Rv3425
The Rv3425 protein was cloned and expressed as previously
described.
15
The ORF encoding the Rv3425 protein was amplified by
PCR which contained 10 ng of M. tuberculosis H37Rv genomic DNA as
template in a total volume of 50
m
lwith2.0UofTaq polymerase
(Promega, Shanghai, PR China). The primers and parameters for
thermal cycle amplification are shown in Table 1 . The amplicons
comprising the full-length Rv3425 ORFs were cloned at the BamHI
and HindIII sites of the cloning vector pUC18. The clones were
confirmed by sequencing, using the T7 promoter primer, on a Prism
377 DNA sequencer (Applied Biosystems, Warrington, UK) and were
then sub-cloned into the expression vector pET32a (Invitrogen, Gro-
ningen, The Netherlands)withsix N-terminalhistidinesequence tags.
The construct generated was then transformed into the Escherichia
coli BL21-CodonPlus (DE3)-RP strain (Novagen, Madison, WI, USA) for
expression. The over-expressed His-tagged recombinant Rv3425
protein was purified from the insoluble inclusion bodies contained in
1 l of isopropyl-b-
D
-thiogalactopyranoside-induced batch cultures by
affinity chromatography using a His-Bind Column (Novagen, USA) in
the presence of 8 M urea, according to the manufacturer’s recom-
mendations. The purified recombinant protein was dialysed against
20 mM Tris–HCl, pH 7.5, containing 100 mM NaCl and glycerol 3%
(v/v). Then, the recombinant protein purity and content were
assessed by SDS-PAGE analysis and Bradford’s assay, respectively.
Finally, the recombinant protein was confirmed by HPLC–MS analysis
Agilent, USA.
2.3. Multiple-antigen ELISA system
The optimal concentration of each antigen, dilution of sera and
dilution of labeled antihuman-IgG (goat) conjugate for ELISA were
determined according to the Phalanx titration principle.
Table 1
PCR primers and thermal cycle parameters for amplification of open reading frame of Rv3425.
Primer Sequence PCR parameters Amplicon size
Forward GAC
GGA TCC ATG CAT CCA ATG ATA CCA GC
*
94
C for 5 min;
Then 30 cycles:
94
C for 45 s;
62
C for45 s;
72
C for 45 s;
Then 72
C for 10 min.
528 bp
Reverse GAC
AAG CTT CTA CCC GCC CCT GTA GAT CTG
y
*
BamHI recognition sequence is underlined.
y
HindIII recognition sequence is underlined.
S.-L. Zhang et al. / Tuberculosis 89 (2009) 278–284 279
Page 2
Combinations which gave the highest signal–noise ratios (S/N)were
determined as optimum. And the signal–noise ratios were deter-
mined by the ratio of the OD values of the positive control sera to
those of the negative control sera at each combination. Polystyrene
flat-bottomed microtiter plates (Costar, USA) were coated with
either of the antigens with a concentration of 2
m
g/ml Rv3425, 5
m
g/
ml recombinant protein 38 kDa (ImmunoDiagnostics, Inc., USA) and
10
m
g/ml LAM (Jiangyin Bio-science, Inc., PR China) diluted in
0.05 M carbonate–bicarbonate buffer (pH 9.6). The plates were
incubated overnight at 4
C and washed three times with washing
buffer (PBST) containing 0.05% Tween-20. After blocking the plates
for 2 h at 37
C with 3% bovine serum albumin (BSA) in PBST
(pH 7.4), the plates were washed again three times and 100
m
lof
serum (diluted 1:100 in buffer PBST, pH 7.4) was added to each well.
All the samples were tested in duplicate. The plates were incubated
for 1 h at 37
C, washed three times with washing buffer, and filled
with 100
m
l of a 1:5000 dilution of antihuman-IgG horseradish
peroxidase (Sigma, Poole, UK). Then, the plates were incubated for
1hat37
C in a water bath and washed again five times, followed by
the addition of 100
m
l of TMB substrate solution (0.04% TMB, 0.04%
urea-peroxide in 0.1 M sodium acetate–citric acid buffer, pH 4.0).
After 10-min incubation in the dark at room temperature, the
reaction was stopped by the addition of 50
m
lof2MH
2
SO
4
to each
well, and the OD
450
was measured with a microtiter plate reader
(Bio-kinetics Reader, Bio-tech Instruments, Winooski, USA).
2.4. Commercial testing kits
Two commercially available kits for the detection of TB antibody,
TB-DOT (Upper Bio-tech, Shanghai, PR China) and TB-Check-1
(Veda.Lab, France), were used to evaluate our multiple-antigen
ELISA system for serodiagnosis of TB by using the same serum
samples. Both of these kits are licensed for clinical use and have
been widely used for clinical assistant diagnosis and screening of
TB in health and clinical settings in PR China. The serum samples
processing and antibody assays were performed according to the
manufacturer’s instructions.
2.5. Statistical analysis
For statistical analysis, the mean OD value and standard devia-
tion (SD) were calculated using the Statistics Package for Social
Science 13.0 (SPSS Inc., Chicago, IL, USA). Sensitivity was defined as
the percentage of individuals in the true-positive group who
showed OD values higher than the cut-off value. Specificity was
defined as the percentage of individuals in the true-negative group
who showed ELISA values that were lower than the cut-off value.
The evaluation of positive sera for multiple-antigen ELISA was
based on a positive score derived from the OD values above the cut-
off point. The cut-off value was calculated from the mean OD plus
two or three standard deviations (SD) from the healthy control
group. The serum tested was determined to be TB positive
according to the criteria as follows: (1) any two or three antigens
specifically react with serum when the cut-off value was calculated
from the mean OD plus two standard deviations; (2) any antigen
specifically reacts with serum when the cut-off value was calcu-
lated from the mean OD plus three standard deviations.
3. Results
3.1. ORF Rv3425 was expressed as a His-tagged fusion
protein in E. coli
The corresponding gene was amplified and cloned and
confirmed by sequencing. The expression vector pET32a-Rv3425
was constructed and expressed in E. coli BL21-CodonPlus (DE3)-
RP cells. Recombinant Rv3425 protein was purified as a 6 His-
tagged fusion protein and fractionated by electrophoresis on
a 12% polyacrylamide gel. A single band corresponding to about
37.5 kDa (including the mass of N-terminal fusion domain of
pET32a) protein was observed on staining the gel with Coomassie
brilliant blue dye (Figure 1). The recombinant protein was largely
present in the insoluble fraction, and therefore the purification
was carried out under denaturing conditions from insoluble
fractions in the presence of 8 M urea, with a yield of 6.5 mg of
protein/l of culture. The over-expressed N-terminal His-tagged
Rv3425 protein was purified to >98% homogeneity on a nickel
affinity column. The purified recombinant Rv3425 protein was
confirmed by HPLC–MS analysis (Figure 2) and used for immu-
noreactivity analyses.
3.2. ELISA assays for IgG antibodies against Rv3425, 38 kDa
and LAM antigens, alone or in combination
The serum IgG antibody responses to the Rv3425, 38 kDa and
LAM antigens, alone or in combination, were measured by ELISA in
patients with active TB and healthy controls (Table 2). Of the
59 smear-positive TB patients, serum samples from 45.8%
of patients had antibodies to Rv3425, serum samples from 37.3% of
patients had antibodies to 38 kDa, and serum samples from 55.9%
of patients had antibodies to the LAM antigen. In the cohort of 62
smear-negative TB patients, 22.6% cases had anti-Rv3425, 9.7%
cases had anti-38 kDa, and 19.4% cases had anti-LAM antibodies. Of
the 15 extra-pulmonary TB patients, no serum sample was reactive
with the 38 kDa antigen, only two (13.3%) serum samples were
reactive with the Rv3425 antigen, and three (20%) serum samples
were reactive with the LAM antigen.
By combining the reactivity with Rv3425, 38 kDa and LAM
antigens, the sensitivity of antibody detection rose to 89.8% in
smear-positive TB, to 43.5% in smear-negative TB, and to 26.7% in
extra-pulmonary TB.
Compared with the total sensitivity estimates provided by the
individual antigens (Rv3425 31.6%, 38 kDa 20.6%, LAM 35.3%),
the total sensitivity achieved by a combination of Rv3425, 38 kDa
and LAM antigens was higher, 61.8%.
Figure 1. Expression and purification of the recombinant PPE protein Rv3425. The
recombinant protein was expressed in strain BL21-CodonPlus (DE3)-RP of E. coli and
was purified to homogeneity using the His-bind column protein purification kit. SDS-
PAGE patterns of total cellular protein from strains with or without the expression
vector were visualized by Coomassie Brilliant Blue R
250
staining. Lanes 1: E. coli strain
BL21-CodonPlus (DE3)-RP without the expression vector. Lane 2: E. coli strain BL21-
CodonPlus (DE3)-RP harboring the expression vector pET32a. Lanes 3–7: E. coli strain
BL21-CodonPlus (DE3)-RP harboring the expression vector pET32a-Rv3425. Lane 8:
purified recombinant protein Rv3425. M: protein molecular weight marker.
S.-L. Zhang et al. / Tuberculosis 89 (2009) 278–284280
Page 3
With respect to the controls, no serum samples from healthy
subjects reacted against Rv3425 antigen, although one serum
sample from them showed antibody to 38 kDa protein, and two
from them showed antibodies to LAM antigen. The specificity
estimate for the multiple-antigen test was high, 93.0%, though
lower than the specificity estimates for the individual antigens
(Table 2).
3.3. Antibody responses of human sera with two diagnostic kits
To evaluate the total (cumulative) sensitivity and specificity of
the combination of the multiple antigens, antibody responses of the
sera from the same group of active TB patients and healthy controls
were analyzed in parallel with TB-DOT and TB-CHECK-1 tests, both
of which are commercially available. The results obtained by the
two kits are summarized in Table 3. The sensitivity of antibody
detection with TB-DOT was 71.2% (42/59) in smear-positive TB,
48.4% (30/62) in smear-negative TB, and 13.3% (2/15) in extra-
pulmonary TB, respectively. TB-CHECK-1 provided a detection
sensitivity of 35.6% (21/59) for smear-positive TB, 30.6% (19/62) for
smear-negative TB, and 20.0% (3/15) for extra-pulmonary TB,
respectively.
The total sensitivity with the TB-DOT test was 51.5% (70/136)
and with that of TB-CHECK-1 test was 31.6% (43/136); and the
specificity with two kits was 73.7% (42/57) and 93.0% (53/57),
respectively (Table 3).
3.4. Comparison of the test accuracy
Accuracies of ELISA assays for IgG antibodies against Rv3425,
38 kDa and LAM antigens, alone or in combination and that of two
commercially available kits in the detection of 136 active TB
patients and 57 healthy controls are shown in Figure 3. The
multiple-antigen ELISA provided the highest accuracy (71.0%)
compared with the single-antigen ELISA (43.5–53.4%) and the
commercial kits (TB-DOT 58.0%; TB-CHECK-1 49.7%).
4. Discussion
During the last few years, advances in molecular techniques,
such as genome sequencing, cloning, expression and purification of
proteins, have accelerated the identification of novel antigens for
the serodiagnosis of TB, and a number of novel antigens are being
tested individually or in combinations to obtain the desired sensi-
tivity and specificity.
12,23–25
However, there is a long way to go to
develop an efficient serodiagnostic tool for TB because infection
with M. tuberculosis passes through several stages with different
composition of antibodies. Any single M. tuberculosis antigen is not
enough to be used to cover the antibody profiles of active
x10
5
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
+ESI Scan (13.623-14.550 min, 57 scans) Frag=200.0V fd01.d Subtract Deconvoluted (Isotope Width=11.8)
37412.57
37453.88 37626.38
Counts vs. Deconvoluted Mass
(
amu
)
36850 36900 36950 37000 37050 37100 37150 37200 37250 37300 37350 37400 37450 37500 37550 37600 37650
Figure 2. MS analysis was performed with ABI165 HPLC–MS (PerkinElmer, USA). The purified recombinant protein Rv3425 only showed a major peak at w37.5 kDa.
Table 2
Sensitivities and specificities of ELISA assays for IgG antibodies against Rv3425, 38 kDa and LAM antigens, alone or in combination.
Sensitivity
*
Specificity
y
Smear-positive TB (n ¼ 59) Smear-negative TB (n ¼ 62) Extra-pulmonary TB (n ¼ 15) Total of TB (n ¼ 136) Healthy controls (n ¼ 57)
Rv3425
x
45.8% (27/59) 22.6% (14/62) 13.3% (2/15) 31.6% (43/136) 100% (57/57)
38 kDa
x
37.3% (22/59) 9.7% (6/62) 0% (0/15) 20.6% (28/136) 98.2% (56/57)
LAM
x
55.9% (33/59) 19.4% (12/62) 20% (3/15) 35.3% (48/136) 96.5% (55/57)
Rv3425 þ 38 kDa þ LAM
**
89.8% (53/59) 43.5% (27/62) 26.7% (4/15) 61.8% (84/136) 93.0% (53/57)
*
Sensitivity was determined by dividing the number of positive cases by the total number of TB patients.
y
Specificity was determined by dividing the number of negative controls by the total number of healthy controls.
x
The cut-off value was calculated from the mean OD value plus two standard deviations (SD) for healthy controls.
**
The serum tested was determined to be TB positive according to the criteria as follows: (1) any two or three antigens specifically react with serum when the cut-off value
was calculated from the mean OD plus two standard deviations; (2) any antigen specifically reacts with serum when the cut-off value was calculated from the mean OD plus
three standard deviations.
S.-L. Zhang et al. / Tuberculosis 89 (2009) 278–284 281
Page 4
TB patients, but combinations of antigens may yield improved level
of sensitivity, without affecting specificity.
26–29
To date, there are
still no commercially available serodiagnostic tests for active
TB with acceptable sensitivity and specificity for routine laboratory
use.
30
Accordingly, it is necessary to investigate novel specific
antigens and optimal combinations of antigens for serodiagnosis of
active TB.
Our previous study has described a protein antigen of M.
tuberculosis, Rv3425, which is a member of the PPE family of pro-
teins, encoded by an open reading frame (ORF) found in RD11 of M.
tuberculosis.
15
Further study showed that Rv3425 protein has
a strong immunogenicity and may be a novel candidate antigen for
the serodiagnosis of active TB; 38 kDa protein located in the plasma
membrane or secreted outside the cell of M. tuberculosis is a phos-
phate-transport lipoprotein. Many research results have revealed
that 38 kDa protein is an immunodominant antigen of M. tuber-
culosis that could help distinguish between healthy bacille Calm-
ette–Guerin (BCG)-vaccinated controls, other mycobacteria and
TB patients.
31,32
Recently, it was also reported that 38 kDa was
associated with active TB.
25
Lipoarabinomannan (LAM) is a major
component of M. tuberculosis cell wall antigen with a molecular
weight of 17.5 kDa,
33,34
and it is composed of glycosyl phosphatidyl
inositol (GPI), mannoses (Mans), Arabinoses (Aras) and caps
(Caps).
35–37
As an important immunomodulating substance with
strong immunogenicity and a good specificity, LAM antigen is
widely used for the serodiagnosis of TB.
38
In the present study, the immunodiagnostic potential of the
Rv3425 antigen has been further evaluated in terms of its humoral
immunoreactivity by assessing IgG antibody levels in the sera of
136 active TB patients and 57 healthy controls. Then the cumulative
sensitivity and specificity of the combination of the multiple anti-
gens, including Rv3425, 38 kDa and LAM, were evaluated with two
commercialized kits.
Our results revealed that Rv3425 antigen is a potential candi-
date for the serodiagnosis of active TB. Of 59 sera from smear-
positive TB patients, 27 (45.8%) had antibodies to Rv3425. Rv3425
antigen provided a sensitivity of 45.8%, lower than that (55.9%) of
LAM antigen, but higher than that (37.3%) of 38 kDa antigen.
Likewise, among a total of the 136 active TB patients, Rv3425
antigen provided a detection sensitivity of 31.6%, lower than that
(35.3%) of LAM antigen, but higher than that (20.6%) of 38 kDa
antigen, with an overall specificity of 100% (57/57).
Furthermore, among the most problematic cases in terms of
diagnosis (smear-negative pulmonary TB and extra-pulmonary
TB patients), though antibodies were detectable in only a small
proportion of patients (from 0% to 22.6%) with individual anti-
gens, cumulative antibody responses achieved with all three
antigens raised the sensitivity to 43.5% for smear-negative
pulmonary TB, and to 26.7% for extra-pulmonary TB patients.
This is a significant improvement over the sensitivities obtained
for the same cohort with TB-DOT kit (smear-negative pulmonary
TB, 4 8.8% and extra-pulmonary TB, 13.3%) and TB-CHECK-1
(smear-negative pulmonary TB, 30.6% and extra-pulmonary TB,
20.0%), representing an improvement over AFB smear-based
diagnosis.
This present study also showed that the heterogeneous anti-
body response to different antigens could be utilized to increase the
sensitivity, providing the foundation for the development of
a multiple-antigen system for screening of active TB. Of the
136 active TB patients, the total detection sensitivity of individual
antigen, Rv3425, 38 kDa, and LAM, was 31.6%, 20.6%, and 35.3%,
respectively. By use of a combination of the three antigens, a total
test sensitivity of 61.8% for active TB patients was achieved, while
good specificity (93.0%) remained.
Compared with the two commercially available kits, the
multiple-antigen test offered a sensitivity of 61.8% for active TB
patients, higher than those of TB-DOT (51.5%) and TB-CHECK-1
(31.6%); and it showed a specificity of 93.0%, equal to that of
TB-CHECK-1 (93.0%), but higher than that of TB-DOT (73.7%).
Moreover, the multiple-antigen ELISA test showed the highest
accuracy (71%), far more than that of single-antigen ELISA
(43.5–53.4%) and that of the two commercially available kits
TB-DOT (58.0%) and TB-CHECK-1 (49.7%), thus representing
a promising candidate to be used in public health and clinical
settings for screening of active TB.
The most likely explanation of differences of sensitivity and
specificity between multiple-antigen and two commercially avail-
able kits is that different types of antigens and different combina-
tions of antigens were used for the serodiagnosis of active TB. In
addition, an important factor for the evaluation of sensitivity and
specificity of the multiple-antigen test is determining the cut-off
value for ELISA. In the present study, the cut-off value for single
antigen-based ELISA was calculated from the mean OD plus two
standard deviations from the healthy control group. For multiple-
antigen-based ELISA, the cut-off value was adjusted and the serum
tested was determined to be TB positive according to the criteria as
follows: (1) any two or three antigens specifically react with serum
when the cut-off value was calculated from the mean OD plus two
standard deviations; (2) any antigen specifically reacts with serum
when the cut-off value was calculated from the mean OD plus three
standard deviations. This optimized cut-off value resulted in an
increased sensitivity (61.8%) together with a good specificity
(93.0%).
Table 3
Results for two commercially available kits.
Sensitivity
*
Specificity
y
Smear-positive TB (n ¼ 59) Smear-negative TB (n ¼ 62) Extra-pulmonary TB (n ¼ 15) Total of TB (n ¼ 136) Healthy controls (n ¼ 57)
TB-DOT 71.2% (42/59) 48.4% (30/62) 13.3% (2/15) 51.5% (70/136) 73.7% (42/57)
TB-CHECK-1 35.6% (21/59) 30.6% (19/62) 20.0% (3/15) 31.6% (43/136) 93.0% (53/57)
*
Sensitivity was determined by dividing the number of positive cases by the total number of TB patients.
y
Specificity was determined by dividing the number of negative controls by the total number of healthy controls.
0
10
20
30
40
50
60
70
80
Rv3425 38kDa LAM Multi-antigen TB-DOT TB-CHECK-1
Accuracy (%)
Figure 3. Comparison of accuracies of ELISA assays for IgG antibodies against Rv3425,
38 kDa and LAM, alone or in combination, and two existing commercially available kits
in the serodiagnosis of TB. The accuracy was defined as true-positive and true-negative
test results divided by the total number of specimens examined.
S.-L. Zhang et al. / Tuberculosis 89 (2009) 278–284282
Page 5
In studies of humoral immune responses to TB, the consensus is
that single antigen-based assays do not deliver satisfactory sero-
diagnostic performance. Combination tests that use several anti-
gens may be more useful for the development of diagnostic
antibody tests because of the large inter-individual variation in
serological responses. Many previous studies of new serodiagnostic
tests based on the well-known antigens have shown that combi-
nation of the specific antigen from M. tuberculosis gave higher
sensitivity than the individual antigen for assessing patients with
active TB,
39,40
although not all the combinations always confer
increased sensitivity.
41–43
This study showed that the sensitivity
was increased with the use of Rv3425, LAM plus the 38 kDa
antigen, and partly supported the above previous findings.
The present study has several limitations. First, serum samples
from healthy control subjects were all collected from Hebei Inter-
national Traveling Hygiene Health Center. Lack of the sera from
patients in whom pulmonary TB was initially suspected but who
were later found to have nontuberculous respiratory disease may
result in overestimate specificity of the multiple-antigen assay.
Second, clinical laboratory technicians performed TB-DOT and
TB-CHECK-1 tests not in a double blind manner, meaning they were
not blinded to patient diagnosis or results of the culture. However,
the multiple-antigen ELISA assays were conducted in a blinded way
by our investigators who were blinded to diagnosis and study
group. It would be better if all the tests were carried out in a blinded
way.
In this study, our data suggest that the sensitivity and accuracy
of ELISA test based on the combination of Rv3425, 38 kDa and LAM
antigen are superior to those of single antigen-based assays and
those of two existing commercial kits. The higher sensitivity
and specificity, together with the simplicity and cost-effectiveness,
of the novel multiple-antigen ELISA test make it useful for the
diagnosis of smear-negative tuberculosis and of those subgroups of
patients with TB from whom specimens are difficult to obtain i.e.
extra-pulmonary tuberculosis TB and childhood TB. Therefore, it
represents a promising tool for the serodiagnosis and screening of
active TB, particularly in developing countries and some areas with
high prevalence of TB infection and is worthy of further
investigation.
Acknowledgements
This study was supported by China Mega-Projects of Science
Research for the 11th Five Year Plan (no. 2008ZX10003-003), China
Postdoctoral Science Foundation funded project (no.
20 080430624) and Shanghai Commission of Science and Tech-
nology, PR China (no. 08410703800).
Funding: Please see acknowledgements.
Competing interests: None declared.
Ethical approval: Not required.
References
1. WHO. URL: <http://www.who.int/tb/publications/2006/tbhiv_recommendations.
pdf>; 2006.
2. Tiwari RP, Hattikudur NS, Bharmal RN, Kartikeyan S, Deshmukh NM, Bisen PS.
Modern approaches to a rapid diagnosis of tuberculosis: promises and chal-
lenges ahead. Tuberculosis (Edinb) 2007;87:193–201.
3. Young DB, Perkins MD, Duncan K, Barry 3rd CE. Confronting the scientific
obstacles to global control of tuberculosis. J Clin Invest 2008;118:1255–65.
4. Ling DI, Flores LL, Riley LW, Pai M. Commercial nucleic-acid amplification tests
for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-anal-
ysis and meta-regression. PLoS ONE 2008;3:e1536.
5. Menzies D, Pai M, Comstock G. Meta-analysis: new tests for the diagnosis of
latent tuberculosis infection: areas of uncertainty and recommendations for
research. Ann Intern Med 2007;146:340–54.
6. Pai M, Zwerling A, Menzies D. Systematic review: T-cell-based assays for the
diagnosis of latent tuberculosis infection: an update. Ann Intern Med
2008;149:177–84.
7. Ariga H, Harada N. Evolution of IGRA researches. Kekkaku 2008;83:641–52.
8. Porsa E, Cheng L, Graviss EA. Comparison of an ESAT-6/CFP-10 peptide-based
enzyme-linked immunospot assay to a tuberculin skin test for screening of
a population at moderate risk of contracting tuberculosis. Clin Vaccine Immunol
2007;14:714–9.
9. Pai M, Kalantri S, Dheda K. New tools and emerging technologies for the
diagnosis of tuberculosis: part I. Latent tuberculosis. Expert Rev Mol Diagn
2006;6:413–22.
10. Nienhaus A, Schablon A, Diel R. Interferon-gamma release assay for the diag-
nosis of latent TB infection analysis of discordant results, when compared to
the tuberculin skin test. PLoS ONE 2008;3:e2665.
11. Lalvani A, Nagvenkar P, Udwadia Z, Pathan AA, Wilkinson KA, Shastri JS, et al.
Enumeration of T cells specific for RD1-encoded antigens suggests a high
prevalence of latent Mycobacterium tuberculosis infection in healthy urban
Indians. J Infect Dis 2001;183:469–77.
12. Rosenkrands I, Aagaard C, Weldingh K, Brock I, Dziegiel MH, Singh M, et al.
Identification of Rv0222 from RD4 as a novel serodiagnostic target for tuber-
culosis. Tuberculosis (Edinb) 2008;88:335–43.
13. Barth RE, Mudrikova T, Hoepelman AI. Interferon-gamma release assays
(IGRAs) in high-endemic settings: could they play a role in optimizing global TB
diagnostics? Evaluating the possibilities of using IGRAs to diagnose active TB in
a rural African setting. Int J Infect Dis 2008;12:e1–6.
14. Khan N, Alam K, Nair S, Valluri VL, Murthy KJ, Mukhopadhyay S. Association of
strong immune responses to PPE protein Rv1168c with active tuberculosis. Clin
Vaccine Immunol 2008;15:974–80.
15. Zhang H, Wang J, Lei J, Zhang M, Yang Y, Chen Y, et al. PPE protein (Rv3425)
from DNA segment RD11 of Mycobacterium tuberculosis: a potential B-cell
antigen used for serological diagnosis to distinguish vaccinated controls from
tuberculosis patients. Clin Microbiol Infect 2007;13
:139–45.
1
6.
Shen
G, Behera D, Bhalla M, Nadas A, Laal S. Peptide-based antibody detection
for tuberculosis diagnosis. Clin Vaccine Immunol 2009;16:49–54.
17. Lee JS, Jo EK, Noh YK, Shin AR, Shin DM, Son JW, et al. Diagnosis of pulmonary
tuberculosis using MTB12 and 38-kDa antigens. Respirology 2008;13:432–7.
18. Tong M, Jacobi CE, van de Rijke FM, Kuijper S, van de Werken S, Lowary TL, et al.
A multiplexed and miniaturized serological tuberculosis assay identifies anti-
gens that discriminate maximally between TB and non-TB sera. J Immunol
Methods 2005;301:154–63.
19. Khan IH, Ravindran R, Yee J, Ziman M, Lewinsohn DM, Gennaro ML, et al.
Profiling antibodies to Mycobacterium tuberculosis by multiplex microbead
suspension arrays for serodiagnosis of tuberculosis. Clin Vaccine Immunol
2008;15:433–8.
20. Wang J, Qie Y, Zhang H, Zhu B, Xu Y, Liu W, et al. PPE protein (Rv3425) from
DNA segment RD11 of Mycobacterium tuberculosis: a novel immunodominant
antigen of Mycobacterium tuberculosis induces humoral and cellular immune
responses in mice. Microbiol Immunol 2008;52:224–30.
21. Metchock B, Nolte FS, Wallace RJ. Mycobacterium. In: Murray PR, Baron EJ,
Pfaller MA, Tenover FC, Yolken RH, editors. Manual of clinical microbiology. 7th
ed. Washington, DC: ASM Press; 1999. p. 399–437.
22. Zhang SL, Shen JG, Shen GH, Sun ZQ, Xu PH, Peng YL, et al. Use of a novel
multiplex probe array for rapid identification of Mycobacterium species from
clinical isolates. World J Microbiol Biotechnol 2007;23:1779–88.
23. Hendrickson RC, Douglass JF, Reynolds LD, McNeill PD, Carter D, Reed SG, et al.
Mass spectrometric identification of MTB81, a novel serological marker for
tuberculosis. J Clin Microbiol 2000;38:2354–61.
24. Silva VM, Kanaujia G, Gennaro ML, Menzies D. Factors associated with humoral
response to ESAT-6, 38 kDa and 14 kDa in patients with a spectrum of tuber-
culosis. Int J Tuberc Lung Dis 2003;7:478–84.
25. Davidow A, Kanaujia GV, Shi L, Kaviar J, Guo X, Sung N, et al. Antibody profiles
characteristic of Mycobacterium tuberculosis infection state. Infect Immun
2005;73:6846–51.
26. Chan ED, Heifets L, Iseman MD. Immunologic diagnosis of tuberculosis:
a review. Tuber Lung Dis 2000;80:131–40.
27. Fujita Y, Ogata H, Yano I. Clinical evaluation of serodiagnosis of active tuber-
culosis by multiple-antigen ELISA using lipids from Mycobacterium bovis BCG
Tokyo 172. Clin Chem Lab Med 2005;43:1253–62.
28. Abebe F, Holm-Hansen C, Wiker HG, Bjune G. Progress in serodiagnosis of
Mycobacterium tuberculosis infection. Scand J Immunol 2007;66:176–91.
29. Okuda Y, Maekura R, Hirotani A, Kitada S, Yoshimura K, Hiraga T, et al. Rapid
serodiagnosis of active pulmonary
Mycobacterium tuberculosis by
analy
sis
of
results from multiple antigen-specific tests. J Clin Microbiol 20 04;42:
1136–41.
30. Steingart KR, Henry M, Laal S, Hopewell PC, Ramsay A, Menzies D, et al.
Commercial serological antibody detection tests for the diagnosis of pulmonary
tuberculosis: a systematic review. PLoS Med 2007;4:e202.
31. Young D, Kent L, Rees A, Lamb J, Ivanyi J. Immunological activity of a 38-kilo-
dalton protein purified from Mycobacterium tuberculosis. Infect Immun
1986;54:177–83.
32. Rhodes SG, Gavier-Widen D, Buddle BM, Whelan AO, Singh M, Hewinson RG,
et al. Antigen specificity in experimental bovine tuberculosis. Infect Immun
2000;68:2573–8.
33. Boehme C, Molokova E, Minja F, Geis S, Loscher T, Maboko L, et al. Detection of
mycobacterial lipoarabinomannan with an antigen-capture ELISA in
S.-L. Zhang et al. / Tuberculosis 89 (2009) 278–284 283
Page 6
unprocessed urine of Tanzanian patients with suspected tuberculosis. Trans R
Soc Trop Med Hyg 2005;99:893–900.
34. Hamasur B, Kallenius G, Svenson SB. Synthesis and immunologic character-
isation of Mycobacterium tuberculosis lipoarabinomannan specific oligosac-
charide–protein conjugates. Vaccine 1999;17:2853–61.
35. Strohmeier GR, Fenton MJ. Roles of lipoarabinomannan in the pathogenesis of
tuberculosis. Microbes Infect 1999;1:709–17.
36. Nigou J, Puzo G. Relationships between the structure and the roles of lip-
oarabinomannans and related glycoconjugates in tuberculosis pathogenesis.
Front Biosci 1998;3:e149–63.
37. Chatterjee D, Khoo KH. Mycobacterial lipoarabinomannan: an extraordinary lip-
oheteroglycan with profound physiological effects. Glycobiology 1998;8:113–20.
38. Barenholz A, Hovav AH, Fishman Y, Rahav G, Gershoni JM, Bercovier H.
A peptide mimetic of the mycobacterial mannosylated lipoarabinomannan:
characterization and potential applications. J Med Microbiol 2007;56:579–86.
39. Ramalingam B, Uma Devi KR, Raja A. Isotype-specific anti-38 and 27 kDa (mpt
51) response in pulmonary tuberculosis with human immunodeficiency virus
coinfection. Scand J Infect Dis 2003;35:234–9.
40. Steingart KR, Dendukuri N, Henry M, Schiller I, Nahid P, Hopewell PC, et al.
Performance of purified antigens for serodiagnosis of pulmonary tuberculosis:
a meta-analysis. Clin Vaccine Immunol 2009;16:260–76.
41. Julian E, Matas L, Alcaide J, Luquin M. Comparison of antibody responses to
a potential combination of specific glycolipids and proteins for test sensitivity
improvement in tuberculosis serodiagnosis. Clin Diagn Lab Immunol
2004;11:70–6.
42. Steingart KR, Henry M, Laal S, Hopewell PC, Ramsay A, Menzies D, et al.
A systematic review of commercial serological antibody detection tests for the
diagnosis of extrapulmonary tuberculosis. Thorax 2007;62:911–8.
43. Verma RK, Jain A. Antibodies to mycobacterial antigens for diagnosis of
tuberculosis. FEMS Immunol Med Microbiol 2007;51:453–61.
S.-L. Zhang et al. / Tuberculosis 89 (2009) 278–284284
Page 7
  • Source
    • "Our hypothesis relies on the heterogeneous humoral response to different antigens as the cause of the increased specificity of the assay (Zhang et al. 2009), in agreement with current guidelines for the diagnosis of infections involving an antibody response, thus suggesting the importance of having multiple antigens to define the antibody profile related to early diagnosis and follow-up of the specific illness (Steller et al. 2005; Sartain et al. 2006). Although further work is now needed to establish "
    [Show abstract] [Hide abstract] ABSTRACT: Unlabelled: Several serological diagnostics rely on enzyme-linked immunosorbent assay (ELISA) to detect bacterial infections. However, for some pathogens, including Bartonella henselae, diagnosis still depends on manually intensive, time-consuming assays including micro-immunofluorescence, Western blotting or indirect immunofluorescence. For such pathogens, there is obviously still a need to identify antigens to establish a reliable, fast and high-throughput assay (Dupon et al. ). We evaluated two B. henselae proteins to develop a novel serological ELISA: a well-known antigen, the 17-kDa protein, and GroEL, identified during this study by a proteomic approach. When serum IgG were tested, the specificity and sensitivity were 76 and 65·7% for 17-kDa, respectively, and 82 and 42·9% for GroEL, respectively. IgM were found to be more sensitive and specific for both proteins: 17-kDa protein, specificity 86·2% and sensitivity 75%; GroEL, specificity 97·7% and sensitivity 45·3%. IgM antibodies were also measured in lymphoma patients and patients with Mycobacterium tuberculosis infection to assess the usefulness of our ELISA to distinguish them from B. henselae infected patients. The resulting specificities were 89·1 and 93·5% for 17-kDa protein and GroEL, respectively. Combining the results from the two tests, we obtained a sensitivity of 82·8% and a specificity of 83·9%. Our work described and validated a proteomic approach suitable to identify immunogenic proteins useful for developing a serological test of B. henselae infection. Significance and impact of the study: A reliable serological assay for the diagnosis of Cat Scratch Disease (CSD) - a pathological condition caused by Bartonella henselae infection - has not yet been developed. Such an assay would be extremely useful to discriminate between CSD and other pathologies with similar symptoms but different aetiologies, for example lymphoma or tuberculosis. We investigate the use of two B. henselae proteins - GroEL and 17-kDa - to develop a serological-based ELISA, showing promising results with the potential for further development as an effective tool for the differential diagnosing of B. henselae infection.
    Full-text · Article · May 2014 · Letters in Applied Microbiology
  • Source
    • "We have not found LAM to be a particularly good antigen for the study and diagnosis of TB in badgers using the ELISA format (unpublished work). The evaluation of LAM as a serodiagnostic antigen in human TB is also not encouraging, although sensitivity is improved if combined with other antigens (Zhang et al., 2009; Beyene et al., 2010 ). Assessment of different antigens, singly and in combination with animals in a single study, would be valuable. "
    [Show abstract] [Hide abstract] ABSTRACT: The infection of both captive and free-ranging wildlife species with pathogenic mycobacteria (including Mycobacterium tuberculosis) poses a zoonotic risk and continues to cause challenges for the livestock industry, zoos and governments around the world. Central to the management and control of tuberculosis is timely and accurate diagnosis. In many cases, bacterial culture is insufficiently sensitive and confirmation of TB post-mortem is neither feasible nor desirable. In this context, there is still considerable research interest in, and need for, immunological methods for diagnosis. Reviews on this topic were published in 2005 and 2009, but since then veterinarians and other researchers have continued to evaluate immunodiagnostic approaches to TB. These include serological tests such as lateral-flow devices, and enzyme-linked immunosorbent assay (ELISA) and those based on evaluation of cell-mediated immunity, such as the tuberculin skin test and interferon-gamma release assay (IGRA). Since 2009, the range of publications on this topic has been extended to a number of new species, including South American camelids, black rhinoceros, lions and non-human primates. Therefore, it seemed appropriate to review the literature in the 3 years since 2009 and provide an overview of progress.
    Full-text · Article · Nov 2013 · Transboundary and Emerging Diseases
  • Source
    • "Indeed, among 49 potential diagnostic proteins for melioidosis reported by Felger et al., more than a half were < 60 kDa [34]. Smaller molecular weight proteins have also been reported as sensitive diagnostic antigens for tuberculosis [25], acute leptospirosis [35] and Brucella abortus infec- tion [36]. In summary, the present study demonstrated that TssD-5 is a potentially superior diagnostic candidate for melioidosis. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Burkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of disease are diverse, ranging from chronic infection to acute septicaemia. The current gold standard of diagnosis involves bacterial culture and identification which is time consuming and often too late for early medical intervention. Hence, rapid diagnosis of melioidosis is crucial for the successful management of melioidosis. Methods The study evaluated 4 purified B. pseudomallei recombinant proteins (TssD-5, Omp3, smBpF4 and Omp85) as potential diagnostic agents for melioidosis. A total of 68 sera samples from Malaysian melioidosis patients were screened for the presence of specific antibodies towards these proteins using enzyme-linked immunosorbent assay (ELISA). Sera from patients with various bacterial and viral infections but negative for B. pseudomallei, as well as sera from healthy individuals, were also included as non-melioidosis controls. The Mann Whitney test was performed to compare the statistical differences between melioidosis patients and the non-melioidosis controls. Results TssD-5 demonstrated the highest sensitivity of 71% followed by Omp3 (59%), smBpF4 (41%) and Omp85 (19%). All 4 antigens showed equally high specificity (89-96%). A cocktail of all 4 antigens resulted in slightly reduced sensitivity of 65% but improved specificity (99%). Multiple-antigen ELISA provided improved sensitivity of 88.2% whilst retaining good specificity (96%). There was minimal reactivity with sera from healthy individuals proposing the utility of these antigens to demarcate diseased from non-symptomatic individuals in an endemic country. Conclusions TssD-5 demonstrated high detection sensitivity and specificity and the results were obtained within a few hours compared to time consuming culture and IFAT methods commonly used in a clinical setting. The use of multiple-antigens resulted in improved sensitivity (88.2%) whilst maintaining superior specificity. These data highlight the use of TssD-5 and other recombinant antigens tested in this study as potential serodiagnostic agents for melioidosis.
    Full-text · Article · Apr 2013 · BMC Infectious Diseases
Show more