Making a differential diagnosis between tuberculous and non-tuberculous pleural effusions represents a critical clinical problem. Conventional methods for diagnosis of pleural TB have proven to be inadequate.[24
This study confirms that ex vivo pleural fluid INF-γ level is an accurate marker for the diagnosis of pleural TB. Also, it showed that QFT-IT assay in peripheral blood or its adapted versions using pleural fluid and/or washed pleural fluid cells had no diagnostic advantage over pleural fluid INF-γ in the diagnosis of pleural TB.
Several previous studies demonstrated that ADA, INF-γ, IL-12p40, IL-18, IAP, and sIL-2R levels were significantly higher in tuberculous than in non-tuberculous pleural effusions;[10
] INF- γ is the most sensitive and specific indicator of tuberculous pleuritis among these six biological markers.[18
] The high specificity of INF-γ is based on the activity of MTB-specific effector T cells at the inflammatory site; INF-γ is produced by T lymphocytes in response to stimulation by specific antigens or nonspecific antigens and is capable of modifying the response of other cells to the immune system.[25
] INF-γ is known to activate macrophages so that they increase their bactericidal capacity against MTB. Therefore, INF-γ levels in pleural fluid may reflect the stimulation of T lymphocytes by tuberculous antigens.[26
] Intriguingly, in TB patients, these T cells seem to keep on producing high levels of INF-γ in the absence of added TB antigen.[27
] Presumably, this is due to a highly activated state that does not require ongoing antigenic stimulation. Alternatively, the accessory cells present in the pleural fluid may already be fully loaded with TB antigen.[28
Previous studies of the value of INF-γ in diagnosing pleural TB revealed a mean sensitivity of 89% and a specificity of 97%.[29
] Another study by Chegou et al
] denoted that ex vivo
INF-γ has 100% accuracy in diagnosing TB pleuritis. The same promising result was found in this study, where pleural fluid INF-γ levels accurately differentiated between all TB and non-TB effusions with 100% sensitivity and specificity.
The clinical value of all surrogate markers for active TB depends on the pre-test probability.[6
] In high clinical probability settings, the use of ADA ≥ 50 U/l makes additional tests unnecessary in a high proportion of patients.[3
] However, ADA as a single marker performed relatively poorly as mentioned in Chegou et al
] study, probably due to the high proportion of non-TB pleural infections, which are characterized by neutrophil-predominant effusions and are a well-known source of false-positive ADA results. In contrast, in the same study[3
] and that of Jiang et al
] a similar high diagnostic accuracy for ex vivo
pleural fluid INF-γ levels in pleural TB was obtained. Also, Hiraki et al
] directly compared six markers including those of ADA, IL-12, p40, IL-18, IAP, sIL-2R, and INF-γ where INF-γ is the most sensitive and specific indicator of tuberculous pleuritis among these six biological markers.
Although determination of INF-γ at the onset of pleural effusion is informative for the diagnosis of tuberculous pleuritis, INF-γ levels have not been used as a routine diagnostic test in suspected TB pleuritis, which may be due to the fact that INF-γ ELISA tests are relatively expensive.[30
In place of the TST test in vivo
, the QFT-TB test in vitro
, an IGRA using ESAT-6 and CFP-10 antigens for the detection of MTB infection was first used commercially for patients with TB infection in Japan in April, 2005.[31
In particular, quantitative results can be obtained within 1 day of blood sampling, and the booster effect caused by sequential skin tests is not an issue, as the assay is performed in vitro
. However, it also has certain limitations. As the assay is based on cellular immunity, its results might depend on the status of the latter. Earlier reports have confirmed low accuracy of the QFT-G in immune-compromised patients.[4
] In addition, the amount of INF-γ produced in this assay was determined by multiple factors, including the number of PBMCs responsible for its production, the time interval between blood sampling and co-incubation with antigens, and the duration of incubation systemic immunosuppression or anergy could cause the inability of TB-specific antigens as well as mitogen to induce INF-γ release[32
] and often leads to the occurrence of the so-called indeterminate results in the QTF Gold assay due to an inability of cells to mount an INF-γ response against the mitogen.[30
] The identification of an appropriate cut-off concentration to fit the objectives is therefore essential.
The usefulness of standardized IGRA in diagnosing active TB disease, especially in high TB incidence areas, is not clear as they do not distinguish latent from active TB disease. The sensitivity of the test in previous studies varied from 55 to 88%, with a mean of 75%.[32
] In this study, the sensitivity obtained with the In Tube version of the blood assay was within this range (70%).
Previous report suggested that the use of pleural effusion mononuclear cells may diagnose pleural TB with a high accuracy (95% sensitivity and 76% specificity). Jafari et al
] used bronchoalveolar lavage-derived mononuclear cells in an INF-γ ELISPOT assay and diagnosed smear-negative pulmonary TB in 37 patients with 100% accuracy, and the technique was superior to the standard PBMC-based ELISPOT assay. Another study by Chegou et al
] reported a similar high diagnostic accuracy (100% sensitivity and 67% specificity) even without MTB-specific antigen stimulation. Our data suggest that pleural cell-based INF-γ assays give 100% sensitivity and 87% specificity which were superior to the measurement of the peripheral blood or pleural fluid, and inferior to the direct measurement of ex vivo
pleural fluid INF-γ in pleural TB. Anatomical factors like the confined pleural space which allows the accumulation of cytokines in the fluid may explain why ex vivo
pleural fluid INF-g performs better than antigen-stimulated cells, as well as an end-stage state of activation of immune cells present in the pleural space.
Beside ADA, other tools have been used to diagnose TB effusion in different studies including fluid lymphocyte/neutrophil ratio, histology and culture of pleural biopsy taken blindly or via thoracoscopy. In this study, we did not measure ADA and not all patients were subjected to pleural biopsy; however, in the study done by Diacon et al
] they concluded that the combination of ADA with the differential pleural fluid WBC count increases specificity. Medical thoracoscopy had the highest diagnostic accuracy with 100% on histology and 76% positive cultures. Of note was the very good result of the combined approach with pleural fluid ADA and lymphocytes: neutrophils plus closed needle biopsy histology and culture, which came very close to thoracoscopy.
The 100% accuracy obtained in the present study is in keeping with other studies.[3
] However, INF-γ levels have not gained widespread acceptance as a routine diagnostic test in suspected TB pleuritis, which may be due to the fact that INF-γ ELISA tests are relatively expensive and that there is a discrepancy between high prevalence of TB pleuritis and available healthcare resources. The cost is nearly tripled if QTF-IT-based tests are used; the most expensive test of them is QTF-IT in pleural fluid cells. However, based on the results of this study, we recommend use of INF-γ ELISA with ex vivo
pleural fluid. It would be both sufficient and more cost effective than pleural fluid or peripheral blood QTF tests and can be performed with small numbers of samples or even single samples by using ELISA.
The strength of this study is the prospective comparison of INF-γ ELISA and three different QTF-based diagnostic tests all applied to the same subjects, who had clinically suspected TB pleural effusion. However, the weak points include the limited number of patients and lack of comparison of these tests with other biomarkers due to limited resources.
In conclusion, INF-γ ELISA with ex vivo pleural fluid would be both sufficient and more cost effective than pleural fluid or peripheral blood QTF-G In tube tests as the use of the QTF test based on either blood or pleural fluid offers no advantage over ex vivo pleural fluid INF-γ measurement in the diagnosis of pleural TB, which should be considered as the test of choice. We have adapted the commercial QTF test to a promising in vitro application using inflammatory cells from the site of disease instead of blood. Such stimulated IGRA using cells from the site of disease are therefore feasible and suggest that new applications should be investigated further in forms of TB that are difficult to diagnose, including smear and culture-negative pulmonary TB, peritonitis, and pericarditis, where aspirated cells could be cultured in the presence of mycobacterial antigens.