Editorial Respiration 2008;76:128–130 DOI: 10.1159/000135933
Can Pleural Tuberculosis Be Diagnosed Using Interferon-Gamma Release Assays? Rajnish Joshi a, b Madhukar Pai c a
Department of Medicine, Mahatma Gandhi Institute of Medical Sciences, Sevagram, India; Division of Epidemiology, School of Public Health, University of California, Berkeley, Calif., USA; c Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Que., Canada b
The diagnosis of pleural tuberculosis (tubercular pleuritis or TBP) is difficult and continues to pose clinical challenges. Safety of pleurocentesis was established in the late 19th century, and within a few decades, we knew that tuberculous pleural fluid was rich in lymphocytes but poor in acid-fast bacilli [1]. Subsequently, the 2 major advances in diagnosis of TBP have been pleural biopsies (their utility was established in the 1950s, and it remains a reference standard to date) [2] and estimation of adenosine deaminase (ADA) in pleural fluid, which emerged 3 decades later [3]. The high accuracy of ADA in the diagnosis of TBP, with pooled sensitivity and specificity estimates from a meta-analysis of 92% (95% CI 56–100) and 89% (95% CI 55–100), respectively [4], was matched a decade later by free (unstimulated) pleural fluid interferon␥ (IFN-␥) assay, with pooled sensitivity and specificity estimates from a meta-analysis of 89% (95% CI 87–91) and 97% (95% CI 96–98), respectively [5]. Both ADA and IFN-␥ are nonspecific markers of inflammation [6], and despite strong evidence on their accuracy, cost and technical considerations such as lack of simple, standardized kits limit their clinical use in a majority of patients with TBP, especially in developing countries. A recent advance in the diagnosis of latent TB infection (LTBI) has been the development of IFN-␥ release assays (IGRAs), based on the principle that T cells of individuals sensitized with TB antigens produce IFN-␥ when they re-encounter mycobacterial antigens [7]. Cur© 2008 S. Karger AG, Basel 0025–7931/08/0762–0128$24.50/0 Fax +41 61 306 12 34 E-Mail
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rently available IGRAs use antigens specific to Mycobacterium tuberculosis, such as the early secreted antigenic target 6 and culture filtrate protein 10. Two commercial kits are now available, the T-SPOT.TB쏐 (Oxford Immunotec, Oxford, UK) and QuantiFERON-TB쏐 Gold In Tube (Cellestis Ltd., Carnegie, Vic., Australia). IGRAs have primarily been designed to detect LTBI using peripheral T cells from blood samples, and there is considerable evidence about their high specificity for LTBI [8, 9]. Two unconventional applications of these tests have been attempted, firstly, in the diagnosis of active TB disease using peripheral blood T cells, and secondly, in the diagnosis of extrapulmonary TB using T cells from the disease site. Both approaches have had mixed results. The sensitivity of IGRAs is not perfect (approximately 80%) in patients with culture-confirmed TB disease [8, 10], and there is no clear trend for change in IFN-␥ levels in patients with active pulmonary TB during the course of TB treatment [11]. Furthermore, IGRAs cannot reliably distinguish between LTBI and active TB disease. Less than perfect sensitivity of IGRA at the time of TB diagnosis could either be due to diminished cellular immunity (i.e. anergy) or due to sequestration of TB-specific T cells to the site of active disease. In patients with TBP, pleural fluid is available with relative ease, and hence, it is a convenient model to evaluate and compare antigen-specific T-cell responses from the site of active disease (i.e. pleura) versus peripheral cirRajnish Joshi, MD, MPH Department of Medicine Mahatma Gandhi Institute of Medical Sciences Sevagram 442102 (India) Tel. +91 9881 018 559, Fax +91 7152 284 967, E-Mail
[email protected]
Table 1. Studies of IGRA on pleural fluid and peripheral blood samples in patients with TBP Studies
Country
Studies from low TB incidence countries Wilkinson et al. [16], 2005 UK Japan Ariga et al. [12], 2007a Losi et al. [14], 2007 Italy, Germany, Netherlands
IGRA
MTB antigens
Pleural fluid IGRA positivity number positive/total
Peripheral blood IGRA positivity number positive/total
ELISPOT QFT-G ELISPOT
ESAT-6 ESAT-6, CFP-10 ESAT-6, CFP-10
8/8 (100) 27/28 (96.4) 19/20 (95.0)
7/7 (100) 21/27 (77.7) 18/20 (90.0)
ESAT-6, CFP-10 ESAT-6, CFP-10, TB7.7 ESAT-6, CFP-10, TB7.7
8/18 (44.4) 12/27 (44.4) 13/23 (56.5)
12/20 (60.0) 17/24 (70.8) 16/22 (72.7)
Studies from intermediate and high TB incidence countries Sohn et al. [15], 2005 Korea QFT-G South Africa QFT-G In-Tube Baba et al. [13], 2008b Chegou et al. [18], 2008 South Africa QFT-G In-Tube
Figures in parentheses are percentages. MTB = Mycobacterium tuberculosis; ELISPOT = enzyme-linked immunospot assay; ESAT-6 = early secreted antigenic target 6; QFT-G = QuantiFERON-TB Gold assay; CFP-10 = culture filtrate protein 10. a Two of the 28 patients in this study had nonpleural tubercular serositis. b Of all patients with TB, 82% were HIV positive; 14/27 (52%) of pleural fluid and 6/24 (25%) of peripheral blood results were indeterminate.
culation (i.e. blood). Such comparisons can help define the immunology of extrapulmonary TB and enable the assessment of the accuracy of pleural fluid IGRA as a diagnostic test for TBP. Of the 5 previous studies [12–16] that performed IGRA on peripheral blood as well as pleural fluid samples, 4 [12–14, 16] reported a higher absolute T-cell response in pleural fluid specimens as compared to the peripheral blood. The pleural fluid cellular response to early secreted antigenic target 6 and culture filtrate protein 10 was measured to be 4.2- to 7.5-fold higher than responses from peripheral blood mononuclear cells [14]. These studies support the argument that in active TB disease, M. tuberculosis specific T cells compartmentalize to the site of disease and contribute to local host defense mechanisms [17]. However, the utility of IGRA as a diagnostic test for TBP remains controversial. While 3 studies [12, 14, 16] from low TB incidence countries reported a high pleural fluid IGRA positivity in patients with TBP, 3 other studies [13, 15, 18] from intermediate and high TB incidence countries have reported relatively lower IGRA positivity rates (table 1). Studies that reported a high IGRA positivity rate in pleural fluid samples have advocated the diagnostic utility of these tests, whereas studies that found lower IGRA positivity have suggested that the conventional biomarkers (such as ADA and unstimulated IFN␥) may be more useful.
There are several unresolved issues involved in the use of IGRAs on pleural fluid samples. Firstly, given the likely variation in the number of T cells in pleural fluid specimens, the first challenge is to determine how much pleural fluid volume and antigen concentration is necessary for performing IGRAs in a reproducible manner. This is particularly relevant for the QuantiFERON-TB Gold In Tube assay, which is designed for whole-blood samples. Secondly, background IFN-␥ levels are known to be high in TB pleural fluid. The degree of baseline stimulation of T cells may be directly proportional to the antigen load, which is likely to be higher in patients in high TB incidence countries. This may result in a blunting of T-cell response in some individuals, and hence, a negative IGRA result or indeterminate results due to high response in the negative controls. Thirdly, although the cut-off value for the peripheral blood IGRA response is defined, the cutoff value for the pleural fluid IGRA is not yet defined. Different authors have used various cut-off levels derived from small studies in heterogeneous settings. This will pose problems for routine clinical use. Larger, comparable studies are required to determine the appropriate cutoff levels for positivity, which may need to be different in high versus low TB incidence countries. It may be hypothesized that pleural fluid IGRA would have a lower specificity in high TB incidence settings, due to high background prevalence of latent TB. This ties in
TBP Diagnosis Using IGRA
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with the known fact that IGRAs cannot distinguish between TB disease and latent infection. Work is ongoing to identify newer antigens and biomarkers that may reliably distinguish between LTBI and active disease [6]. The study by Chegou et al. [18] in this issue of Respiration underscores the need to determine the diagnostic accuracy of pleural fluid IGRAs in a larger representative sample in high TB incidence countries, especially in populations with a high prevalence of HIV infection (which is known to affect IGRA performance). To be clinically useful, fu-
ture studies must demonstrate that IGRAs are more accurate and cost-effective than already validated nonspecific biomarkers. Acknowledgments R.J. is recipient of a training fellowship from the Fogarty AIDS International Training Program (grant 1-D43-TW00003-17), USA. M.P. is supported by a CIHR New Investigator Award.
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