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(Chest. 2003;123:740-744.)
© 2003 American College of Chest Physicians

Diagnostic Significance of Interferon-{gamma} in Tuberculous Pleural Effusions*

Keisuke Aoe, MD, PhD; Akio Hiraki, MD, PhD; Tomoyuki Murakami, MD, PhD; Ryosuke Eda, MD, PhD; Tadashi Maeda, MD, PhD; Kazuro Sugi, MD, PhD and Hiroyasu Takeyama, MD, PhD

* From the Department of Respiratory Medicine (Drs. Aoe, Hiraki, Eda, Maeda, and Takeyama) and Clinical Research (Drs. Murakami and Sugi), National Sanyo Hospital, Respiratory Disease Center, Yamaguchi, Japan.

Correspondence to: Keisuke Aoe, MD, PhD, Department of Respiratory Medicine and Clinical Research, National Sanyo Hospital, Respiratory Disease Center, 685 Higashi-Kiwa, Ube, Yamaguchi 755-0241, Japan; e-mail: keisukeaoe{at}mtf.biglobe.ne.jp


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Tuberculosis (TB), the single most frequent infectious cause of death worldwide, also is a major cause of pleural effusion, which in TB usually has lymphocytic and exudative characteristics. Differential diagnosis between TB and nontuberculous pleural effusion can be sometimes difficult, representing a critically important clinical problem.

Methods: We studied 46 patients presenting with pleural effusion to the National Sanyo Hospital between April 2000 and January 2001 (34 men and 12 women; mean age, 64 years). Ten patients (22%) had tuberculous pleurisy, 19 patients (41%) had malignant pleuritis, and 17 patients (37%) had pleural effusion due to an etiology other than tuberculosis or cancer. Pleural fluid concentrations of four suggested markers were measured using commercially available kits.

Results: The pleural fluid levels (mean ± SE) of adenosine deaminase (83.3 ± 18.2 U/L vs 25.8 ± 20.4 U/L, p < 0.0001), interferon-{gamma} (137 ± 230 IU/mL vs 0.41 ± 0.05 IU/mL, p < 0.0001), immunosuppressive acidic protein (741 ± 213 µg/mL vs 445 ± 180 µg/mL, p < 0.001) and soluble interleukin 2 receptor (7,618 ± 3,662 U/mL vs 2,222 ± 1,027 U/mL, p < 0.0001) were significantly higher for tuberculous pleuritis than for other causes of effusion. Receiver operating characteristic analysis demonstrated that pleural fluid content INF-{gamma} was the best indicator of tuberculous pleurisy among four relevant biological markers.

Conclusions: INF-{gamma} in pleural fluid is the most sensitive and specific among four biological markers for tuberculous pleuritis. Thus, our results suggest that determination of INF-{gamma} at the onset of pleural effusion is informative for the diagnosis of tuberculous pleuritis. Further studies including larger numbers of patients are needed to verify this result.

Key Words: adenosine deaminase • immunosuppressive acidic protein • interferon-{gamma} • pleural fluid • soluble interleukin 2 receptor • tuberculous pleuritis


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Worldwide, tuberculosis (TB) is the single most frequent cause of death by an infectious agent.1 TB also is a major cause of pleural effusion; tuberculous effusions usually are lymphocytic and exudative. The diagnosis of tuberculous pleuritis should be considered in any patient with an exudative pleural effusion. Management of patients with tuberculous pleuritis who have acquired pleural effusion requires an effective treatment plan based on timely and accurate diagnostic information.

The diagnosis of tuberculous pleuritis commonly is made from observation of granulomas in pleural biopsy specimens or a culture finding positive for Mycobacterium tuberculosis from pleural tissue or pleural fluid. However, sensitivity of these methods is sufficiently low that even when histopathology and culture are combined, the diagnosis can be uncertain or missed in "negative" cases.2 3 While repeating invasive diagnostic procedures ultimately may yield positive results, such an approach places patients at increased risk of complications and also increases costs. A reliable clinical marker providing physicians with rapid and accurate diagnosis of tuberculous pleuritis is greatly needed.

A variety of biological markers have been proposed to aid in the diagnosis of tuberculous pleuritis, including increased pleural fluid concentrations of adenosine deaminase (ADA),4 5 6 interferon (INF)-{gamma},7 8 9 10 11 12 13 immunosuppressive acidic protein (IAP),14 15 and soluble interleukin 2 receptors (sIL-2Rs).16 17 18 19 20 However, which of these markers is most useful for diagnosis of tuberculous pleuritis has not been determined. Here we studied 46 patients with pleural effusion to determine whether ADA, INF-{gamma}, IAP, and sIL-2R concentrations in pleural fluid show associations with the cause of pleural effusion.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Forty-six inpatients presenting with pleural effusions who were admitted to National Sanyo Hospital between April 2000 and January 2001 were studied. Informed consent was obtained according to the guidelines of good clinical practice in Japan. Clinical signs and symptoms, demographic data, and radiologic results were recorded. Of these 34 men and 12 women (mean age, 64 years), 10 patients had tuberculous pleurisy (22%), 19 patients had malignant pleuritis (41%, including 16 patients with lung cancer, 2 patients with malignant pleural mesothelioma, and 1 patient with cancer of the uterocervix) and 17 patients had pleural effusions with various nontuberculous, nonmalignant etiologies (37%; pneumonia in 4 patients, rheumatoid arthritis in 2 patients, heart failure in 2 patients, renal failure in 1 patient, and undetermined etiology in 8 patients).

Specimen Collection and Processing
For each subject, at least 40 mL of pleural fluid was collected in a syringe during thoracentesis. A portion of the sample was submitted for acid-fast staining, bacteriologic examination, cytologic examination, and measurement of protein, albumin, total bilirubin, lactate dehydrogenase (LDH), and glucose. Another part of the sample was centrifuged at 2,000 revolutions per minute for 10 min. The supernatant was frozen at - 80° until assays for markers.

For mycobacterial culture, each sample was inoculated in duplicate on Ogawa medium and assessed weekly for mycobacterial growth for a total of 8 weeks. TB was identified by a positive niacin test result.

Diagnosis of Tuberculous, Malignant, and Miscellaneous Pleural Effusions
Patients received a diagnosis of tuberculous effusion when they met any the following criteria: TB isolated from pleural fluid or pleural tissue; granulomas in the pleural tissue showing staining for acid-fast bacilli; or granulomas in pleural tissue that did not stain for acid-fast bacilli, but showed a response to antituberculous treatment; or a sputum culture finding positive for TB in the presence of a pleural effusion.

Malignant pleural effusions were diagnosed either by a positive pleural fluid cytologic result or malignant cells identified in a pleural biopsy specimen. In addition, even when both of these test results were negative, malignant effusion was diagnosed when a primary cancer was known to have disseminated and pleural fluid concentrations of tumor markers were elevated. Miscellaneous pleural effusions were those not linked to tuberculosis or malignant disease as defined above.

Determination of Pleural Fluid Levels of ADA, INF-{gamma}, IAP, and sIL-2R
ADA activity was measured by autoanalyzer using commercially available kits (Nitobo Medical; Tokyo Japan). INF-{gamma} was measured using commercially available enzyme-linked immunosorbent assay kits (Medical and Biological Laboratories; Nagoya, Japan). IAP activity was measured by autoanalyzer using commercially available kits (Sanko-Junyaku; Tokyo, Japan), while sIL-2R was measured using commercially available enzyme-linked immunosorbent assay kits (Yamanouchi; Tokyo, Japan).

Statistical Analysis
Differences in pleural fluid characteristics and biological markers between tuberculous and nontuberculous pleural effusions, or between tuberculous, malignant, and miscellaneous effusions, were examined by the Mann-Whitney U test. To compare performances of markers, receiver operating characteristic (ROC) curves were constructed.21


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pleural Fluid Characteristics
Pleural fluid concentrations of protein, albumin, total bilirubin, lactate dehydrogenase (LDH), cholesterol, and glucose are shown in Table 1 . Protein, albumin, and cholesterol concentrations were significantly higher in tuberculous effusions than in nontuberculous pleural effusions. Glucose concentrations were significant lower in tuberculous effusions compared with nontuberculous pleural effusions. No differences in total bilirubin and LDH concentrations were noted between groups.


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  		Table 1.. Pleural Fluid Characteristics in Tuberculous and Nontuberculous Pleural Effusions*

 
ADA, INF-{gamma}, IAP, and sIL-2R in Pleural Fluid
As shown in Figure 1 , TB patients exhibited higher pleural fluid ADA concentrations than non-TB patients (83.3 ± 18.2 U/L vs 25.8 ± 20.4 U/L, p < 0.0001; Fig 1 , top left, A). TB patients also exhibited higher pleural fluid concentrations (mean ± SE) of INF-{gamma} (37 ± 230 IU/mL vs 0.41 ± 0.05 IU/mL, p < 0.0001) [top right, B], IAP (741 ± 213 µg/mL vs 445 ± 180 µg/mL, p < 0.001) [bottom left, C], and sIL-2R (7,618 ± 3,662 U/mL vs 2,222 ± 1,027 U/mL, p < 0.0001) [bottom right, D] than non-TB patients.



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  		Figure 1.. The plural fluid concentrations of IAP (top left, A), INF-{gamma} (top right, B), sIL-2R (bottom left, C), and ADA (bottom right, D) in TB and non-TB patients.

 
ROC Analysis
To determine the most sensitive and specific indicator for diagnosis of tuberculous pleuritis among the four biological markers, we performed ROC analysis (Fig 2 ) that clearly demonstrated that pleural fluid INF-{gamma} was the most useful diagnostic indicator for tuberculous pleurisy. The second best test was sIL-2R.



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  		Figure 2.. ROC analysis. Pleural fluid INF-{gamma} concentrations are more useful for distinguishing tuberculous pleurisy compared with ADA, IAP, and sIL-2R.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Differential diagnosis between tuberculous and nontuberculous pleural effusions represents a critical clinical problem. Conventional methods for diagnosis of pleural tuberculosis have proven insufficient. Direct examination of pleural fluid by Ziehl-Neelsen staining requires bacillar densities of 10,000/mL and, therefore, has low sensitivity (0 to 1%).2 3 Although culture is more sensitive (11 to 50%),22 23 2 to 6 weeks are required to grow M tuberculosis, and a minimum of 10 to 100 viable bacilli are needed. The sensitivity of pleural biopsy reportedly is higher than thoracentesis whether in terms of culture (39% vs 79%)2 22 or histologic evaluation (71 to 80%).2 3 However, biopsy requires greater expertise, is more invasive, and is subject to sampling error.

Many biological markers measured in pleural effusions have been proposed to aid diagnostic sensitivity for tuberculous pleuritis, including ADA, IFN-{gamma}, IAP, and sIL-2R. Several previous reports demonstrated that these four markers were significantly higher in tuberculous than in nontuberculous pleural effusions,4 5 6 7 8 9 10 11 12 13 14 15 and our results confirmed this. Whatever roles individual marker may play in development of pleural effusion, our results suggest that the pleural fluid ADA, IAP, sIL-2R, and IFN-{gamma} are useful indicators for diagnosis of tuberculous pleuritis.

To date, no reports have directly compared performance of multiple markers such as ADA, IFN-{gamma}, IAP, or sIL-2R. Therefore, we carried out ROC analysis to determine the most sensitive and specific indicator of tuberculous pleuritis among these four markers, which demonstrated that INF-{gamma} was the most sensitive and specific indicator in the differential diagnosis of tuberculous pleuritis. However, further studies including larger numbers of patients are needed to verify this result. In addition, results of testing for these biological markers should be compared with polymerase chain reaction findings in pleural fluids.

INF-{gamma} is an important immune regulator that exhibits both antiviral and cytotoxic activities.24 25 INF-{gamma} is produced by T lymphocytes in response to stimulation with specific antigens or nonspecific antigens, and is capable of modifying the response of other cells to the immune system. INF-{gamma} is known to activate macrophages, increasing their bactericidal capacity against M tuberculosis. Therefore, INF-{gamma} detected in pleural fluid may be the result from stimulation of T lymphocytes by tuberculous antigens. As we have found INF-{gamma} to be the most sensitive and specific indicator for diagnosis of tuberculous pleuritis, an INF-{gamma} assay may prove to be an important screening test for tuberculous pleuritis. Nevertheless, measurement of INF-{gamma} is relatively expensive compared with ADA assays. Although the cost of a test can be an important consideration, especially in less-developed countries with a high incidence of disease, our results suggests that INF-{gamma} concentrations in pleural fluid should be determined routinely in patients strongly suspected to have TB.

In conclusion, our results demonstrate that the pleural fluid concentrations of ADA, INF-{gamma}, IAP and sIL-2R in patients with tuberculous pleural effusions are significantly higher than in other effusions. Most importantly, ROC analysis clearly demonstrated INF-{gamma} to be the most sensitive and specific among four relevant biological markers for diagnosis of tuberculous pleuritis. Thus, our results suggest that determination of INF-{gamma} at the onset of pleural effusion is informative for the diagnosis of tuberculous pleuritis. Further studies including larger numbers of patients should be undertaken confirm this result.


   Acknowledgements
 
We thank Drs. M. Moriyama, H. Kohara, K. Makihata, K. Takao, and K. Murakami for providing clinical samples and critical comments.


   Footnotes
 
Abbreviations: ADA = adenosine deaminase; IAP = immunosuppressive acidic protein; INF = interferon; LDH = lactate dehydrogenase; ROC = receiver operating characteristic; sIL-2R = soluble interleukin 2 receptor; TB = tuberculosis

Received for publication April 30, 2002. Accepted for publication September 12, 2002.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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Chest Online, 20 Mar 2003 [Full text]
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