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  Table of Contents  
Year : 2017  |  Volume : 35  |  Issue : 2  |  Page : 290-292

Levels of circulating immune complexes containing Mycobacterium Tuberculosis-specific antigens in pulmonary tuberculosis and sarcoidosis patients

1 Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Pulmonary Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Date of Web Publication5-Jul-2017

Correspondence Address:
I Verma
Department of Biochemistry, PGIMER, Chandigarh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.209569

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 ~ Abstract 

The present study was conducted to understand the aetiological link between tuberculosis (TB) and sarcoidosis. Sera from smear-positive TB subjects (n = 24), smear-negative TB subjects (n = 24), sarcoidosis patients (n = 24) and healthy controls (n = 24) were collected and circulating immune complexes were isolated. Sandwich ELISA was performed for detecting four highly specific mycobacterial regions of difference (RD) proteins (early secretory antigenic target 6 [ESAT6], 10 KDa culture filtrate protein [CFP10], 21 KDa CFP [CFP21] and mycobacterial protein from species TB [MPT 64]). Sensitivity and specificity was calculated, and receiver operating characteristic plots were plotted. Non-parametric Mann–Whitney U-test was used to calculate statistical significance. Seventy per cent of sarcoidosis patients showed the presence of immune complexes of mycobacterial RD proteins similar to that observed in the sera of smear-negative TB patients as opposed to antibody-based detection assay based on these RD proteins. Thus, immunoassays based on specific mycobacterial RD proteins also need to be developed and validated carefully to differentiate TB and sarcoidosis, a close mimic of smear-negative tuberculosis.

Keywords: Circulating immune complexes, pulmonary tuberculosis, regions of differences antigens, sandwich ELISA, sarcoidosis

How to cite this article:
Goyal B, Sheikh J A, Agarwal R, Verma I. Levels of circulating immune complexes containing Mycobacterium Tuberculosis-specific antigens in pulmonary tuberculosis and sarcoidosis patients. Indian J Med Microbiol 2017;35:290-2

How to cite this URL:
Goyal B, Sheikh J A, Agarwal R, Verma I. Levels of circulating immune complexes containing Mycobacterium Tuberculosis-specific antigens in pulmonary tuberculosis and sarcoidosis patients. Indian J Med Microbiol [serial online] 2017 [cited 2018 Feb 20];35:290-2. Available from:

 ~ Introduction Top

Sarcoidosis is an idiopathic disorder characterised by multisystem non-caseating granulomas. Numerous genetic, autoimmune and infectious mechanisms have been described to play a role. There has always been a concern of Mycobacterium tuberculosis or its antigens to play an aetiological role in sarcoidosis due to similar clinical presentation, radiological features and histopathological picture depicting granulomas.[1],[2],[3],[4] In fact, it has been hypothesised that genetically distinct individuals respond differently to mycobacterial infection leading to either tuberculosis (TB) or sarcoidosis.[5] A recent meta-analysis has revealed the presence of mycobacterial DNA in around 30% of sarcoidosis patients.[6] Studies carried out in our laboratory also demonstrated mycobacterial DNA in 50% sarcoidosis patients using polymerase chain reaction for heat shock protein 65 (hsp65)[7] and presence of antibodies to specific mycobacterial regions of difference (RD) proteins such as early secretory antigenic target 6 (ESAT 6)/10 kDa culture filtrate antigen [CFP 10] in the sera of sarcoidosis patients.[8] RDs are genomic regions that are present in M. tuberculosis and are deleted in most of the Bacillus Calmette- Guerin strains and other non-TB mycobacteria. Recently, it has been reported from our laboratory that antibodies to bioinformatically selected immunodominant epitopes of four RD antigens including ESAT 6 and CFP 10 of RD 1 along with 21 kDa CFP (CFP 21) and mycobacterial protein from species TB 64 (MPT 64) of RD 2 could be specifically detected in pulmonary TB (PTB) patients and not in sarcoidosis patients.[9] These studies indicated that in spite of mycobacterial aetiology of sarcoidosis, carefully designed immunoassays can be used to differentiate the two diseases. Apart from antibody detection, antigen detection too in serum has earlier been employed for diagnosis of TB.[10],[11] The present study is another attempt in this direction to study the role of mycobacterial RD antigen detection in the circulating immune complexes (CICs) of PTB and sarcoidosis patients.

 ~ Materials and Methods Top

A case–control, comparative group study was conducted at a tertiary care hospital in Northern India. Sarcoidosis (n = 24) patients with clinical features, radiological evidence (chest radiograph/high resolution computed tomography scan) suggestive of sarcoidosis or non-caseating granuloma on transbronchial biopsy were enrolled for the study. All sarcoidosis patients were negative for tuberculin skin test with 1 tuberculin unit purified protein derivative. Smear-positive TB subjects (n = 24) were recruited from directly observed treatment short-course centre of our institute. Smear-negative TB subjects (n = 24) included patients with clinical or radiological evidence of PTB that were sputum smear-negative and culture-positive. Healthy volunteers (n = 24) were also included in the study. Venous blood samples were collected from all the subjects after obtaining written informed consent. Serum was separated and stored at 80°C until use. The study was approved by the Institute Ethics Committee.

Polyethylene glycol (2.5%) was used to precipitate the CICs as described elsewhere.[12] Briefly, 150 μl serum sample was mixed with veronal-buffered saline (VBS, pH - 7.6), incubated overnight at 4°C and centrifuged at 2000 ×g for 20 min at 4°C. The precipitates were washed and dissolved in 2.5% VBS. Sandwich ELISA was carried out for detecting four M. tuberculosis specific antigens simultaneously in the precipitated immune complexes. Polyclonal sera raised in mice immunised with the combination of ESAT6, CFP 10, CFP 21 and MPT 64 using dimethyldioctadecylammonium liposomes with monophosphoryl lipid-A as adjuvants were used as a capture antibody in the pre-standardised dilution of 1:3000. Plates were incubated overnight at 4°C and blocked with 3% bovine serum albumin for 1 h. Optimally diluted CICs (1:40) were added in each well and incubated for 2 h at 37°C. Horseradish peroxidase-conjugated anti-human IgG (1:10,000 dilution) was added to each well for 90 min. O-Phenylenediamine was then added as substrate. The reaction was stopped after 15–20 min by adding sulphuric acid, and optical density (OD) was read at 492 nm. Each experiment was carried out in triplicates at different time points to ensure reproducibility, and the sample showing reactivity in at least two of the three assays was considered positive.

Statistical analysis

The percentage recognition was determined taking mean OD plus two times the standard deviation of control samples as cut-off. Sensitivity and specificity was calculated for ELISA tests in different diagnostic categories using Microsoft Excel™. Non-parametric Mann–Whitney U-test was used to determine statistical significance between different categories using GraphPad Prism v5 (GraphPad Software, San Diego, CA, USA) and dot plots were plotted. P < 0.05 was considered statistically significant. The Receiver Operating Characteristics (ROC) plots using SPSS (Version 16, SPSS Inc, Chicago, IL) for MS-Windows were plotted and area under curve (AUC) was calculated.

 ~ Results and Discussion Top

The antigen detection assay based on RD antigens showed the sensitivity of 91.6% and 70.8% in smear-positive and smear-negative TB patients, respectively. However, RD antigens in immune complexes from the sera of sarcoidosis patients also showed reactivity of 70.8%, same as that of smear-negative TB patients [Figure 1]. The ROC curve of smear-positive and smear-negative TB patients versus healthy control subjects showed AUC 0.979 and 0.994, respectively, indicating that test has high specificity and discriminatory power to distinguish PTB patients from healthy controls. However, smear-positive and smear-negative TB patients versus sarcoidosis showed AUC 0.624, 0.612 indicating some overlap in PTB and sarcoidosis patients group.
Figure 1: The dot plot and receiver operating characteristic curve depicting the reactivity of sera for circulating immune complexes in smear-positive, smear-negative, sarcoidosis and healthy control group and area under curve of these groups, respectively. *Signifies statistical significant difference between different groups at P < 0.05. The receiver operating characteristic curve of smear-positive and smear-negative tuberculosis patients versus control showed area under curve 0.979 and 0.994, respectively. However, smear-positive and smear-negative tuberculosis patients versus sarcoidosis showed area under curve 0.624, 0.612, respectively, indicating that test has high specificity and discriminatory power to distinguish pulmonary tuberculosis patients from healthy controls, but low area under curve against sarcoidosis indicates some overlap in pulmonary tuberculosis and sarcoidosis patients group.

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These results clearly showed the presence of mycobacterial RD antigens in 70% of sarcoidosis patients. These findings further validate the previous studies wherein the presence of mycobacterial DNA and antibodies to mycobacterial RD antigens has been demonstrated in sarcoidosis patients.[6],[7],[8] This is the first report to demonstrate the presence of M. tuberculosis-specific antigens in the sarcoid patients as compared to earlier observation of various Hsps that are highly cross-reactive. These findings further uphold the mycobacterial aetiology of sarcoidosis. The results of present study are in contrast to earlier reports wherein B-cell response to in silico-derived specific epitopes of RD antigens could differentiate both PTB and sarcoidosis patients [9] that could be attributed to the increased specificity of short peptides and epitopes. Further studies are needed to validate the utility of different epitopes for their differential diagnostic potential and elucidate whether the two diseases are just the manifestation of different epitope presentation with a common aetiological link. Based on these findings as well as earlier studies from our laboratory and considering the mycobacterial aetiology of sarcoidosis and cross-reactivity of certain mycobacterial epitopes in sarcoidosis, it can be concluded that the immunoassays to differentiate TB and sarcoidosis involving both antibody as well as antigen detection assays should be carefully designed particularly using the specific B-cell epitopes of M. tuberculosis complex-specific antigens such as RD antigens.


The fellowship of BG by ICMR, New Delhi, is acknowledged.

Financial support and sponsorship

ICMR provided the funds for this study to BG under the ICMR scheme for MD/MS-PhD programme.

Conflicts of interest

There are no conflicts of interest.

 ~ References Top

Carlisle J, Evans W, Hajizadeh R, Nadaf M, Shepherd B, Ott RD, et al. Multiple Mycobacterium antigens induce interferon-gamma production from sarcoidosis peripheral blood mononuclear cells. Clin Exp Immunol 2007;150:460-8.  Back to cited text no. 1
Drake WP, Dhason MS, Nadaf M, Shepherd BE, Vadivelu S, Hajizadeh R, et al. Cellular recognition of Mycobacterium tuberculosis ESAT-6 and KatG peptides in systemic sarcoidosis. Infect Immun 2007;75:527-30.  Back to cited text no. 2
Inui N, Suda T, Chida K. Use of the QuantiFERON-TB gold test in Japanese patients with sarcoidosis. Respir Med 2008;102:313-5.  Back to cited text no. 3
Oswald-Richter KA, Culver DA, Hawkins C, Hajizadeh R, Abraham S, Shepherd BE, et al. Cellular responses to mycobacterial antigens are present in bronchoalveolar lavage fluid used in the diagnosis of sarcoidosis. Infect Immun 2009;77:3740-8.  Back to cited text no. 4
Dubaniewicz A. Mycobacterium tuberculosis heat shock proteins and autoimmunity in sarcoidosis. Autoimmun Rev 2010;9:419-24.  Back to cited text no. 5
Gupta D, Agarwal R, Aggarwal AN, Jindal SK. Molecular evidence for the role of mycobacteria in sarcoidosis: A meta-analysis. Eur Respir J 2007;30:508-16.  Back to cited text no. 6
Mootha VK, Agarwal R, Aggarwal AN, Gupta D, Ahmed J, Verma I, et al. The sarcoid-tuberculosis link: Evidence from a high TB prevalence country. J Infect 2010;60:501-3.  Back to cited text no. 7
Agarwal R, Gupta D, Srinivas R, Verma I, Aggarwal AN, Laal S. Analysis of humoral responses to proteins encoded by region of difference 1 of Mycobacterium tuberculosis in sarcoidosis in a high tuberculosis prevalence country. Indian J Med Res 2012;135:920-3.  Back to cited text no. 8
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Goyal B, Kumar K, Gupta D, Agarwal R, Latawa R, Sheikh JA, et al. Utility of B-cell epitopes based peptides of RD1 and RD2 antigens for immunodiagnosis of pulmonary tuberculosis. Diagn Microbiol Infect Dis 2014;78:391-7.  Back to cited text no. 9
Pereira Arias-Bouda LM, Nguyen LN, Ho LM, Kuijper S, Jansen HM, Kolk AH. Development of antigen detection assay for diagnosis of tuberculosis using sputum samples. J Clin Microbiol 2000;38:2278-83.  Back to cited text no. 10
Kalra M, Khuller GK, Grover A, Behera D, Wanchu A, Verma I. Utility of a combination of RD1 and RD2 antigens as a diagnostic marker for tuberculosis. Diagn Microbiol Infect Dis 2010;66:153-61.  Back to cited text no. 11
Kansal R, Singhal R, Khuller GK. Correlation between antibodies to mannophosphoinositides and detection of antigen in circulating immune complexes in patients with pulmonary tuberculosis. Indian J Med Res 1992;95:54-8.  Back to cited text no. 12


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