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ORIGINAL ARTICLE
Year : 2013  |  Volume : 2  |  Issue : 2  |  Page : 92-95

Evaluation of pleural fluid interferon gamma as a diagnostic marker of tuberculous pleural effusion


Government General and Chest Hospital, Osmania Medical College, Hyderabad, India

Date of Web Publication21-May-2013

Correspondence Address:
Jayalakshmi Lingam
G-307, Girisikhara Enclave, Hilltop Colony, Erramanzil, Hyderabad-500082
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2277-8632.112332

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  Abstract 

Context: Establishing etiology of pleural effusion is a common diagnostic problem in clinical practice. The most common cause is tuberculosis (TB). Adenosine deaminase (ADA) is one of the biochemical markers commonly used in the etiological diagnosis of tuberculous pleural effusion. In the past decade several studies on pleural fluid interferon gamma (IFN-γ) showed that it is an important diagnostic marker of TB.
Aim: To evaluate the role of immunological marker IFN-γ in pleural fluid and to compare with pleural fluid ADA in the diagnosis of tuberculous pleural effusion with reference to pleural biopsy histology and treatment outcome.
Materials and Methods: A total of 50 pleural fluid samples with more than 80% lymphocytes were analyzed for IFN-γ by enzyme-linked immunosorbent assay (ELISA) method and the results were compared with pleural fluid ADA. Clinical, radiological, and laboratory evaluation and response to treatment were noted. Among the 50 test samples, 31were from tuberculous pleural effusion, 14 from malignant effusion and five from effusion due to other causes.
Results: Pleural fluid levels of IFN-γ and ADA were significantly higher in tuberculous than in nontuberculous pleural effusions. At the cut off value of 3.7 IU/mL for IFN-γ and 40 U/L for ADA it was found to have sensitivity and specificity of 96.77 and 94.73% for IFN-γ and 83.87 and 78.94% for ADA, respectively; differentiating tuberculous from nontuberculous pleural effusions.
Conclusion: IFN-γ is more sensitive and specific marker than ADA for differentiating tuberculous from nontuberculous pleural effusions and helps in rapid and efficient diagnosis.

Keywords: Adenosine deaminase, interferon gamma, pleural effusion, pleural fluid, tuberculosis


How to cite this article:
Lingam J, Kandi S. Evaluation of pleural fluid interferon gamma as a diagnostic marker of tuberculous pleural effusion. J NTR Univ Health Sci 2013;2:92-5

How to cite this URL:
Lingam J, Kandi S. Evaluation of pleural fluid interferon gamma as a diagnostic marker of tuberculous pleural effusion. J NTR Univ Health Sci [serial online] 2013 [cited 2019 Nov 17];2:92-5. Available from: http://www.jdrntruhs.org/text.asp?2013/2/2/92/112332


  Introduction Top


Pleural effusion is an abnormal accumulation of fluid in the pleural space and can be diagnozed by clinical and radiological features. The two frequent causes of pleural effusion are tuberculosis (TB) and malignancy. Other causes include pneumonia, granulomatous diseases, and pulmonary embolism. [1]

The laboratory tests on pleural fluid which help in etiological diagnosis of pleural effusion include cell count (total and differential count)and cytology,biochemical tests (protein, lactate dehydrogenase, adenosine deaminase (ADA)), and microbiological tests (Ziehl-Neelsen (ZN) stain, culture for aerobic bacteria, fungi,andmycobacteria). [1] Pleural biopsy is performed because the chances of obtaining a diagnosis is much greater than examination of pleural fluid alone but it is more invasive, requires greater expertise, and is subject to sampling error. [2] In case of pleural TB and other paucibacillary forms of the infection, the "gold standard", mycobacterial culture presents significant limitations in sensitivity. Thus, it is advised to interpret alternative diagnostic methods within the context of all laboratory and clinical findings. [2]

Interferon Gamma (IFN-γ) is a cytokine secreted by activated T-lymphocytes usually as part of immune response (T H 1 response). It is regarded as immunoregulatory cytokine and found to have clinical application as an immunostimulator in chronic granulomatous diseases. [3] A delayed hypersensitivity type of response to mycobacterial antigens in pleural space leads to localization of activated T lymphocytes in pleural space and production of large amounts of IFN-γ. [4],[5] It enhances the microbicidal activity of macrophages and promotes differentiation of B cells and CD 8 cells into immunologically effector cells. [2],[5]

Lymphocyte activation leads to elevated levels of ADA. Thus high levels of ADA in pleural fluid is seen in conditions causing effusions with lymphocyte predominance whereas IFN-γ is exclusively seen in patients with verified or suspected TB. [6] Demonstrating high levels of these markers, IFN-γ and ADA in pleural fluid helps in establishing the diagnosis [7] both in immunocompetent and immunocompromised patients. [8]

The objective of the present study is to evaluate the role of immunological marker IFN-γ in pleural fluid in the diagnosis of tuberculous pleural effusion and its comparison with the pleural fluid ADA with reference to pleural biopsy histology and treatment outcome.


  Materials and Methods Top


Fifty pleural fluid samples showing lymphocyte predominance in cytology report with more than 80% lymphocytes received at clinical laboratory of a tertiary hospital during November 2007 to April 2008 were taken into study. Purulent and hemorrhagic samples were excluded from study.

Results of clinical evaluation, chest X-ray, ultrasonography, laboratory tests (CBP, ESR, RBS, HIV screening, sputum for AFB), and tests on pleural fluid (cytology, ZN stain, culture for aerobic bacteria, fungi, and mycobacteria) were noted from the patient's record [Table 1].
Table 1: Results of Laboratory Tests on 50 Pleural Fluid Samples


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The cell-free supernatant of the pleural fluid samples were analyzed for IFN-γ using Immunotech IFN-γ enzyme immunoassay (IM 1743, IM 1862).The sensitivity of this kit is 0.08 IU/mL of IFN-γ in the analyte. This ELISA is a two-step sandwich-type assay and is performed according to the procedure insert of the kit.

Calibration curve was obtained using the results (optical density (OD) values) of the standard concentration sera supplied in the kit. The value of IFN-γ in the test samples was obtained by interpolating the OD value of the test sample on the calibration curve. The cut off value for IFN-γ was identified as 3.7 IU/mL with sensitivity and specificity of 95% by studying 595 patients with pleural effusion. [9]

Pleural fluid ADA levels estimated by colorimetric method were recorded. The cut off used for ADA is 40 U/L. The sensitivity, specificity, and predictive values of both IFN-γ and ADA were prepared by statistical analysis and compared. [10]


  Results Top


Depending on the provisional diagnosis, the 50 pleural fluid test samples were from 31 tuberculous effusions, 14 malignant effusions and five effusions due to other etiology. Out of the 50 samples, 32 were from male patients, ten samples were from HIV reactive cases and of these, seven were tuberculous effusions and three were malignant effusions. One among the seven HIV+TB samples was positive by both ZN stain as well as mycobacterial culture on Lowenstein-Jensen (LJ) medium. Staphylococcus aureus was isolated from one sample among the five effusion cases due to other etiology while in the other four cases the etiology was not established. Fungal culture was negative in all the samples. IFN-γ was above the cut off value in 31 samples and ADA above the cut off value in 30 samples [Table 1].

In tuberculous pleural effusions 96.77% were above cut off, whereas only 5.25% of nontuberculous effusions were showing values above cut off for IFN-γ [Table 2]. Applying standard error of difference in proportions statistically, this difference in positivity for IFN-γ is significant (p-value < 0.001) [Table 3]
Table 2: Values of Ifn-γ and Ada in Pleural Fluid Samples as per the Provisional Diagnosis(n = 50)


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Table 3: Mean value and standard deviation (SD) of ifn-g and ada in the pleural fluid samples (n = 50)


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Pleural fluid IFN-γ in TBcases (n=31):

Value > 3.7 IU/mL = 30

Value < 3.7 IU/mL = 01

Sensitivity = 96.77%

Specificity = 94.73%

Predictive value for positive test = 96.77

Predictive value for negative test = 94.73

P-value for IFN-γ:< 0.001

Pleural fluid ADA in TB cases (n=31):

Value > 40 U/L = 26

Value < 40 U/L = 05

Sensitivity = 83.87%

Specificity = 78.94%

Predictive value for positive test = 84.66

Predictive value for negative test = 75

P-value for ADA: < 0.001

There is significant difference in the mean value of IFN-γ in different conditions in this study group. The mean value of IFN-γ in TB + HIV cases is 16.93 IU/mL and in TB positive and HIV negative cases is 33.4 IU/mL. Both these parameters in the immunocompetent as well as inthe immunocompromised were well above the cut off value indicating the diagnosis of tuberculous pleural effusion. The mean value of IFN-γ in malignant effusions is 0.053 IU/mL and in unknown etiology cases it is 0.007 IU/mL. The present study throws light on the usefulness of IFN-γ estimation in pleural fluid, either for accurate diagnosis of TB or for exclusion.


  Discussion Top


The sensitivity of various investigative methods on pleural fluid for diagnosing TB as given in Davidson's Principles and Practice of Medicine are as follows:

Direct microscopy by ZN stain method = 0-5%

Mycobacterial culture (gold standard) = 25-35%

PCR for Mycobacterium tuberculosis= 80%

Pleural biopsy is highly sensitive when done by thoracoscopy. [1]

The evaluation of the efficiency of alternative diagnostic tests is conducted in reference to the best available standard "gold standard"-mycobacterial culture. In case of pleural TB and other paucibacillary forms of TB, mycobacterial culture presents significant limitations in sensitivity. [2] In order to obtain maximum utility of alternative diagnostic methods, it is important to consider this issue and to interpret results within the context of all laboratory and clinical findings. [2] IFN-γ is an immunoregulatory cytokine [3] secreted in large amounts by activated T lymphocytes in tuberculous pleural effusion cases. [4],[5] High levels of ADA is seen in all pleural effusions with lymphocyte predominance. [6]

It is stated that the diagnosis of tuberculous pleural effusion is established by demonstrating high levels of TB markers like IFN-γ or ADA in pleural fluid, alternatively by culture of pleural fluid or thoracoscopic pleural biopsy. [7] The values of IFN-g were well above the cut off value in tuberculous effusions both in immunocompetent and immunocompromised patients [8] and the cut off value was identified as 3.7 IU/mL. [9] Krenkeet al., [11] in their study on pleural fluid ADA and IFN-g as diagnostic tools in tuberculous pleurisy showed that their estimation helps in definitive diagnosis of TB. Studies by Kim et al., showed that, in lymphocyte predominant pleural effusions estimation of biological markers IFN-g, TNF-∞, IL-2, ADA in pleural fluid help in differentiating tuberculous from malignant pleural effusions. [12],[13] Studies by Valdes et al., and Yung et al., showed that biological markers ADA and IFN-g in pleural fluid appear to be reasonably accurate at detecting tuberculous pleurisy. [14],[15],[16]

The results of the present study correlate with the meta-analysis by Greco et al., which shows the sensitivity and specificity of ADA and IFN-g in pleural fluid for detection of tuberculous pleurisy is 93% for ADA by analyzing 4738 samples and 96% for IFN-γ by analyzing 1189 samples. [17] Sharma and Amit in their study on diagnostic utilityof pleural fluid IFN-γ showed that a cut off value of 138 pg/mL provides the best sensitivity for diagnosis of tuberculous pleural effusion. [18] Sriram et al., showed that cytological analysis of pleural fluid helps in narrowing the differential diagnosis and IFN-γ is highly sensitive and specific marker than ADA in diagnosing TB [19] and similar results were obtained in the present study. Krenke et al., [20] in their study on IFN-γ and ADA measurement in pleural fluid showed that they are as useful as biopsy method in diagnosing tuberculous effusion so could reduce invasive diagnostic procedures. [20]

To summarize, pleural fluid IFN-γ is more sensitive and specific marker with better predictive value for both positive and negative tests than pleural fluid ADA for the etiological diagnosis of pleural effusion.

 
  References Top

1.Crompton GK, McHardy GF. Diseases of the respiratory system. In: Edwards CR, Boucher IA. Davidson's Principles and Practice of Medicine. 16 th ed. Edinburgh:ELBS; 1992. p. 410-2.  Back to cited text no. 1
    
2.Villegas MV, Labrada LA, Saravia NG. Evaluation of polymerase chain reaction, adenosine deaminase, and interferon-gamma in pleural fluid for the differential diagnosis of pleural tuberculosis. Chest 2000;118:1355-64.  Back to cited text no. 2
    
3.Oppenheim JJ, Ruscetti FW. Cytokines. In:Parslow TG, Stites DP, Terr AI, Imboden JB, editors. Medical Immunology. 10 th ed.New York: Lange Medical Books/McGraw Hill Medical Publishing Division; 2001. p. 157-9.  Back to cited text no. 3
    
4.Barnes PF, Fong SJ, Brennan PJ, Twomey PE, Mazumder A, Modlin RL. Local production of tumor necrosis factor and interferon gamma in tuberculouspleuritis. J Immunol 1990;145:149-54.  Back to cited text no. 4
    
5.Ribera E, Ocaña I, Martinez-Vazquez JM,Rossell M, Español T, Ruibal A. High level of interferon gamma in tuberculous pleural effusion. Chest 1998;93:30811.  Back to cited text no. 5
    
6.Söderblom T, Nyberg P, Teppo AM, Klockars M, Riska H,Pettersson T. Pleural fluid interferon-gamma and tumour necrosis factor-alpha in tuberculous and rheumatoid pleurisy. EurRespir J 1996;9:1652-5.  Back to cited text no. 6
    
7.Light RW. Disorders of pleura, mediastinum and diaphragm. In:Braunwald E, Fauci AS, Kasper DL, Hauser SL, Longo DL, Jameson JL.Harrison's Principles of Internal Medicine, vol.2., 15 th ed.New York: McGraw Hill International Edition; 2003:1513-5.  Back to cited text no. 7
    
8.Villena V, López-Encuentra A, Echave-Sustaeta J, Martin-Escribano P, Ortuño-de-Solo B,Estenoz-Alfaro J. Interferon-gamma in 388 immunocompromised and immunocompetent patients for diagnosing pleural tuberculosis. Eur Respir J 1996;9:2635-9.  Back to cited text no. 8
    
9.Villena V, López-Encuentra A, Pozo F, Eschave-Sustaeta J, Ortuño-de-Solo B, Estenoz-Alfaro J, et al. Interferon gamma levels in pleural fluid for the diagnosis of tuberculosis. Am J Med2003;115:365-70.  Back to cited text no. 9
    
10.Park JE, Park K. Health Information and Basic Medical Statistics, Park's Text Book of Preventive and Social Medicine. 21 st ed.Jabalpur: Banarasidas Bhanot Publishers; 2010. p. 778-92.  Back to cited text no. 10
    
11.Krenke R, Safianowska A, Paplinska M, Nasilowski J, Dmowska-Sobstyl B, Bogacka-Zatorska E, et al. Pleural fluid adenosine deaminase and interferon gamma as diagnostic tools in tuberculous pleurisy. J PhysiolPharmacol 2008;59(Suppl 6):349-60.  Back to cited text no. 11
    
12.Kim YC, Park KO, Bom HS, Lim SC, Park HK, Na HJ,et al. Combining ADA, protein and IFN-gamma best allows discrimination between tuberculous and malignant effusion. Korean J Intern Med 1997;12:225-31.  Back to cited text no. 12
    
13.Shimokata K, Saka H, Murate T, Hasegawa Y, Hasegawa T. Cytokine content in pleural effusion, comparison between tuberculous and carcinomatous pleurisy. Chest 1991;99:1103-7.  Back to cited text no. 13
    
14.Valdés L, San José E, Alvarez D, Sarandeses A, Pose A, Chomón B, et al. Diagnosis of tuberculous pleurisy using the biologic parameters adenosine deaminase, lysozyme, and interferon gamma. Chest 1993;103:458-65.  Back to cited text no. 14
    
15.Zhu X, Zhang J, Wu Z. The study of detecting adenosine deaminase, interferon gamma in differentiating tuberculous pleural effusion and neoplastic pleural effusion. Ningxia MedJ 1999;7:1.  Back to cited text no. 15
    
16.Liu YC, Shin-Jung Lee S, Chen YS, Tu HZ, Chen BC, Huang TS. Differential diagnosis of tuberculous and malignant pleurisy using pleural fluid adenosine deaminase and interferon gamma in Taiwan. J Microbiol Immunol Infect 2011;44:88-94.  Back to cited text no. 16
    
17.Greco S, Girardi E, Masciangelo R, Capoccetta G B, Saltini C. Adenosine deaminase and interferon gamma measurements for the diagnosis of tuberculous pleurisy; A meta-analysis. Int J Tuberc Lung Dis 2003;7:777-86.  Back to cited text no. 17
    
18.Sharma SK, Banga A. Diagnostic utility of pleural fluid interferon gamma in tuberculous pleural effusion. J Interferon Cytokine Res 2004;24:213-7.  Back to cited text no. 18
    
19.Sriram PS, Veena B, Antony and M D Kristin A. Pleural effusion. September 2007;47:1-3.  Back to cited text no. 19
    
20.Krenke R, Korczyñski P. Use of pleural fluid levels of adenosine deaminase and interferon gamma in the diagnosis of tuber-culouspleuritis. Curr Opin Pulm Med 2010;16:367-75.  Back to cited text no. 20
    



 
 
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