Enzyme-linked immunospot assay for interferon-gamma in the diagnosis of tuberculous pleurisy

ORIGINAL ARTICLE 10.1111/j.1469-0691.2008.02655.x Enzyme-linked immunospot assay for interferon-gamma in the diagnosis of tuberculous pleurisy L.-N. Lee 1,2, C.-H. Chou 3, J.-Y. Wang 2, H.-L. Hsu 1, T.-H. Tsai 2, I.-S. Jan 1, P.-R. Hsueh 1,2 and P.-C. Yang 2 1) Department of Laboratory Medicine, 2) Department of Internal Medicine, National Taiwan University College of Medicine, National Taiwan University Hospital, Taipei and 3) Department of Internal Medicine, National Taiwan University Hospital Yun-Lin Branch, Yun-Lin, Taiwan Abstract Patients presenting with pleural effusion of undetermined aetiology were prospectively enrolled, and an enzyme-linked immunospot (ELISPOT) assay on pleural fluid and peripheral blood was performed. Forty patients were studied, including 19 with culture- or biopsy-confirmed (n = 15) or clinically compatible (n = 4) tuberculous pleurisy, and 21 with pleural effusions due to non-tuberculous causes. The sensitivity, specificity and positive and negative predictive values of the assay were 94.7%, 85.7%, 85.7% and 94.7%, respectively, on pleural fluid, and 77.8%, 90.5%, 87.5% and 82.6%, respectively, on blood. Antigen-specific, interferon-gamma-secreting T-cells were concentrated eight to ten times in pleural fluid as compared with blood. Among the seven patients not suitable for pleural biopsy and three patients whose biopsy results were non-diagnostic, nine had positive ELISPOT result with pleural fluid. The ELISPOT assay for interferon-gamma can accurately diagnose tuberculous pleurisy and is helpful for patients not suitable for pleural biopsy and those whose biopsy results are non-diagnostic. Keywords: Diagnosis, enzyme-linked immunospot assay, interferon-gamma, tuberculous pleurisy Original Submission: 26 March 2008; Revised Submission: 25 June 2008; Accepted: 25 June 2008 Editor: G. Greub Clin Microbiol Infect 2009; 15: 173 179 Corresponding author and reprint requests: P.-R. Hsueh, Departments of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei 100, Taiwan E-mail: hsporen@ntu.edu.tw Introduction Tuberculous pleurisy is an important cause of pleural effusions, and may account for 14% of all pleural exudates, or 25% of all pleural effusions, in areas with a high incidence of tuberculosis [1 3]. Conventional methods for the diagnosis of tuberculous pleurisy, however, are often inefficient. Biochemical characteristics and cellular components of pleural fluid in tuberculous pleurisy lack specificity [1,4]. The levels of adenosine deaminase (ADA), an enzyme associated with T-lymphocyte activity, and those of interferon-gamma (IFN-c), a cytokine secreted by activated T-cells, have been shown to be increased in, and used to diagnose, tuberculous pleural effusion [5 14]. However, these increases can also be observed in other infectious, inflammatory or malignant diseases [8,11 19], and are not specific for tuberculous pleurisy. The 6-kDa early-secreted antigenic target (ESAT-6) and culture filtrate protein 10 (CFP10) are antigens preferentially found in Mycobacterium tuberculosis and virulent Mycobacterium bovis, but not in M. bovis bacille Calmette Guérin vaccine or most environmental mycobacteria [20 22]. It has been shown that ESAT-6 and CFP10 can stimulate peripheral blood mononuclear cells (PBMCs) from patients with tuberculosis to secrete specific IFN-c. An ex vivo enzyme-linked immunospot (ELISPOT) test employing these antigens and detecting IFN-csecreting T-cells in peripheral blood [23 27] and bronchoalveolar lavage fluid [28] has been found useful in diagnosing latent and active tuberculosis. The potential of T-cells in pleural fluid from patients with tuberculous pleurisy to secrete antigen-specific IFN-c has been studied only rarely [28]. A prospective study was conducted to evaluate ESAT-6- and CFP10-specific IFN-c production by T-lymphocytes in pleural fluid and peripheral blood from patients with pleural effusion of undetermined aetiology, using the ELISPOT assay. The aim was to evaluate the efficiency of this assay in the diagnosis of tuberculous pleurisy. Materials and Methods Patients This prospective study was conducted in a university hospital of 1500 beds in northern Taiwan, and was approved by the Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases

174 Clinical Microbiology and Infection, Volume 15 Number 2, February 2009 CMI Institutional Review Board of the hospital. After written consent had been obtained, patients aged 18 years, with pleural effusion of undetermined aetiology, were enrolled from 1 January to 31 December 2006. Eight millilitres of peripheral blood was drawn into a tube containing sodium citrate, and 10 ml of pleural fluid that had been aspirated during a diagnostic tapping was collected. Standard diagnostic tests for pleural fluid included chemistry, cell count, differential count, cytology, and culture of bacteria, including mycobacteria, and fungi. The ELISPOT assay was performed within 2 h after the collection of blood or pleural fluid. PBMC ELISPOT assay The assay using the ELISPOT kit (T-SPOT-TB; Oxford Immunotec Ltd, Oxford, UK) was performed according to the manufacturer s instructions. PBMCs were separated from whole blood using Ficoll Hypaque, washed, resuspended, and counted. PBMCs (c. 250 000/well) were added to wells coated with a mouse monoclonal anti-ifn-c antibody and containing antigen (ESAT-6 or CFP10), mitogen (phytohaemagglutinin), as positive control, or nothing (background control wells). After 16 18 h of incubation, plates were washed. Fifty microlitres of conjugate reagent containing biotinylated anti-ifn-c monoclonal antibody was added and incubated for 1 h. The plates were then washed, and chromogenic alkaline phosphatase substrate was added. After 7 min, the plates were washed and dried. Spots were counted manually using a hand-held magnifying glass. The number of spots in the background control wells was subtracted from the number in the test wells, and a response was considered positive if the number of spots per test well was 10 (when the background control count was <5), or at least twice the value found in the background control wells (when the background control count was 5). Pleural fluid ELISPOT assay Pleural fluid (6 10 ml) was transferred to a 50-mL Falcon tube. The fluid was diluted with an equal volume of RPMI- 1640 and centrifuged at 430 g for 7 min. The sediment was suspended with 10 ml of RPMI-1640 and centrifuged at 300 g for 7 min. The sediment was then suspended in 0.7 ml of AIM-V culture medium. Forty microlitres of the suspension were mixed with an equal volume of a Trypan blue solution to evaluate the viability of the cells. The T-SPOT-TB assay was performed in each well (100 ll) containing c. 10 3 10 5 cells. The results were validated by counting the spots from the positive control well. Subsequent steps were as stated above for PBMCs. Diagnosis of tuberculous pleurisy Tuberculous pleurisy was classified as confirmed if any of the following criteria were met: (i) culture of pleural fluid, pleural biopsy material or sputum yielded M. tuberculosis; (ii) the histology of pleural biopsy material showed granulomatous inflammation with positive acid-fast bacilli; and (iii) the histology of pleural biopsy material showed granulomatous inflammation without acid-fast bacilli, but there was obvious clinical improvement after antituberculous chemotherapy. Tuberculous pleurisy was classified as probable, if: (i) the culture of pleural fluid, pleural biopsy material or sputum did not yield M. tuberculosis; (ii) the pleural biopsy histology showed chronic inflammation without acid-fast bacilli; (iii) there was obvious clinical improvement after antituberculous chemotherapy; and (iv) there was no evidence of other causes of pleural disease. Clinical improvement was defined as defervescence and disappearance of pleural effusions after antituberculous chemotherapy, without the concomitant use of other antimicrobials or corticosteroids. Diagnosis of pleural effusion due to other causes The diagnosis was based on pleural fluid culture, cytology, histology of pleural or lung biopsy specimens, or compatible clinical, radiographic and nuclear medical features. Statistical analysis Comparisons of the sensitivities and specificities of the PBMC ELISPOT and the pleural fluid ELISPOT assay were performed using Fisher s exact test. Results Patient characteristics During the year 2006, there were 40 patients with undetermined pleural effusion who consented to being tested. Nineteen (15 males) were diagnosed as having tuberculous pleurisy (15 confirmed cases, four probable cases); their average age was 60.5 years (range: 21 102 years). Twentyone (12 males) had a non-tuberculous cause of pleural effusion, with an average age of 65.5 years (range: 25 87 years; p 0.461 compared with tuberculous group). In the tuberculous group, 15 (79%) patients had coexisting diseases, including: end-stage renal disease (n = 2); previous stroke (n = 2); stroke and eosinophilic pneumonitis treated with steroids (n = 1); benign prostatic hyperplasia (BPH) and chronic obstructive pulmonary disease (n = 1); BPH and arrhythmia (n = 1); BPH and hip fracture (n = 1); Parkinson s disease and prostatic cancer (n = 1); senile dementia (n = 1); acute lymphocytic leukaemia; post-bone marrow transplantation

CMI Lee et al. ELISPOT in diagnosing tuberculous pleurisy 175 status (n = 1); diabetes mellitus (n = 1); pneumoconiosis (n = 1); alcoholism and adrenal insufficiency due to use of steroid-containing alternative medicine (n = 1); and normalpressure hydrocephalus with paroxysmal supraventricular tachycardia (n = 1). All had received <2 weeks of antituberculous chemotherapy or were not treated at the time of the ELISPOT study. Pleural fluids of the patients with tuberculous pleurisy were all exudates, and all except two had 76% lymphocytes in the differential count. The 21 control patients had the following diagnoses: pulmonary adenocardinoma (n = 6); small-cell lung cancer (n = 1); metastatic pleural cancer from the colon (n = 1) and breast (n = 1); mesothelioma (n = 1); pleural lymphoma (n = 1); heart failure (n = 3); pulmonary infarction (n = 1); pleurisy due to Mycobacterium avium intracellulare complex (n = 2); cytomegalovirus pneumonia with parapneumonic effusion (n = 1); and pneumonia with parapneumonic effusion (unknown aetiology) (n = 3). Multiple specimens (mean, 8; range, 2 34) from all control patients were culture-negative for mycobacteria. Laboratory tests for human immunodeficiency virus antibodies were performed in five patients in the tuberculous pleurisy group, and in three in the control group. All test results were negative. Of the remaining 32 patients with unknown human immunodeficiency virus status, 30 had their white blood cells counted. Only one had a peripheral blood lymphocyte count <500 (441) 10 9 /L. The patient, a 62- year-old man with a history of alcoholism and adrenal insufficiency, had his lymphocyte count performed after antituberculous therapy. All of the 40 patients were free of AIDSdefining illness [29] before December 2007. Pleural fluid ELISPOT assay The ELISPOT assay was positive in 18 of the 19 patients with tuberculous pleurisy (sensitivity: 94.7%), and in three of the 21 controls (specificity: 85.7%) (Table 1). The only false-negative result occurred in a 78-year-old man with isolation of M. tuberculosis from two specimens of pleural fluid, and coexisting Parkinson s disease and prostate cancer. The three false-positive results occurred in one patient each with mesothelioma, pulmonary adenocarcinoma, and pleurisy due to M. avium intracellulare complex. PBMC ELISPOT assay The PBMC ELISPOT assay was performed for all patients and was successful in all but one with tuberculous pleurisy (the one with the false-negative pleural fluid ELISPOT result). The result was positive in 14 of the 18 patients with tuberculous pleurisy (sensitivity: 77.8%), and in two of the 21 controls (specificity: 90.5%). Table 2 lists the four patients for TABLE 1. Sensitivity and specificity of the ELISPOT assay in the diagnosis of tuberculous pleurisy Specimen tested Cause of pleurisy No. of cases Positive Negative Sensitivity (%) Specificity (%) Positive predictive value Negative predictive value Pleural fluid Confirmed TB (n = 15) 14 1 93.3 Probable TB (n = 4) 4 0 100.0 Total (n = 19) 18 1 94.7 a 85.7 Blood Confirmed TB (n =14 b ) 11 3 78.6 Probable TB (n = 4) 3 1 75.0 Total (n = 18) 14 4 77.8 a 87.5 Pleural fluid Non-TB (n = 21) 3 18 85.7 c 94.7 Blood 2 19 90.5 c 82.6 TB, tuberculosis. a p 0.307. b Peripheral blood mononuclear cells from one patient failed to grow after antigen stimulation in the ELISPOT assay. c p 0.997. TABLE 2. Characteristics of four tuberculous patients with false-negative peripheral blood mononuclear cell ELISPOT assay results Patient, n Age/sex Coexisting disease White blood cell count Lymphocyte (%) Lymphocyte count Anti-HIV 2 62/M Adrenal insufficiency 7600 5.8 441 Not done Alcoholism 7 87/M Arrhythmia 4150 12.8 531 Not done Prostate hyperplasia 13 22/M ALL, 63 days post-bone 3980 14.0 557 marrow transplant 16 87/M Trigeminal neuralgia 8880 5.7 506 Not done Prostate hyperplasia ALL, acute lymphocytic leukaemia; HIV, human immunodeficiency virus.

176 Clinical Microbiology and Infection, Volume 15 Number 2, February 2009 CMI whom the PBMC ELISPOT assay gave a false-negative result. All of them had lymphopenia. Twelve of the 14 patients with a positive PBMC ELISPOT assay result had their white blood cells counted. Their average lymphocyte count was 1071 ± 250/lL (mean ± standard deviation, range: 622 1388), in contrast to 509 ± 50/lL (range: 441 557) for the four lymphopenic patients with a false-negative PBMC ELI- SPOT result (p <0.001). The false-positive result occurred in the patient with mesothelioma whose pleural fluid also gave a positive result, and in one patient with heart failure. Number of immunospots in the pleural fluid and PBMC ELI- SPOT assays In most of the tuberculous patients, the number of immunospots in the pleural fluid ELISPOT assay was much higher than that in the PBMC ELISPOT assay. The median value of pleural fluid/pbmc immunospot ratio was 10 for the ESAT-6 antigen (range: 1 250) and 8 for the CFP10 antigen (range: 0.7 42) (data not shown). Feasibility and sensitivity of the tests for the diagnosis of tuberculous pleurisy Table 3 shows the feasibility and yield rate of various tests used to diagnose tuberculous pleurisy in the patients studied here. Pleural biopsy provided a rapid and correct histological diagnosis in nine of the 12 patients who underwent this procedure, but was not performed in the other seven tuberculous patients, due to: (i) tendency to bleed in three patients (two with end-stage renal disease, and one with acute leukaemia); (ii) dementia and inability to cooperate in two patients; and (iii) refusal by the patient in two cases. Diagnostic tests that require isolation of M. tuberculosis or amplification of its DNA were all suboptimal in sensitivity. TABLE 3. Feasibility and sensitivity of tests used for the diagnosis of tuberculous pleurisy in the patients of this study cdiagnostic test N (positive)/n (total) Sputum acid-fast stain 0/14 0 Pleural fluid acid-fast stain 0/18 0 Pleural biopsy tissue acid-fast stain 3/12 25.0 Bronchial washing acid-fast stain 0/2 0 Sputum culture 7/15 46.7 Pleural fluid culture 8/19 42.1 Pleural biopsy tissue culture 3/8 37.5 Bronchial washing culture 1/2 50.0 Pleural fluid/sputum PCR 0/4 Pleural histology a 9/12 75.0 PBMC ELISPOT assay 14/18 77.8 Pleural fluid ELISPOT assay 18/19 94.7 PBMC, peripheral blood mononuclear cell. a Granulomatous inflammation. Sensitivity c (%) Fig. 1 shows that the pleural fluid ELISPOT assay was positive, and thus very helpful in providing a rapid diagnosis, in nine (90%) of the patients who were either not fit for pleural biopsy (n = 7) or whose pleural histology was non-diagnostic (n = 3). Discussion Our study showed that an ex vivo assay that allows detection of ESAT-6/CFP10 antigen-specific, IFN-c-secreting T-cells in pleural fluid and blood in 24 h was efficient in diagnosing tuberculous pleurisy. Previous studies showed that, with blood samples, the assay had a sensitivity of 87 97% and a specificity of 87 100% for diagnosing bacteriologically confirmed pulmonary and extrapulmonary tuberculosis [23 27]. It has also been used with bronchoalveolar lavage fluid samples and shown to be effective in detecting pulmonary tuberculosis [28]. Regarding its usage in pleural effusion, Wilkinson et al. [30] found that ESAT-6-specific, IFN-csecreting T-cells were concentrated 15-fold in pleural fluid relative to their blood level in ten patients with known tuberculous pleurisy, but were absent in eight patients with known non-tuberculous pleurisy. However, whether this ex vivo assay can be used clinically to diagnose tuberculosis in patients with unknown cause of pleural effusion has not been reported. The present study shows that the ELISPOT assay performed with pleural fluid samples can diagnose tuberculous pleurisy accurately overnight with a sensitivity of 95%. When it was performed with blood samples, the sensitivity was lower, with false-negative results being observed mainly in patients with lymphopenia. Unlike the ELISPOT assay, which detects antigen-specific IFN-c-secreting T-lymphocytes, biochemical or immunological tests, e.g. of ADA and IFN-c in pleural fluids, which have been used to diagnose tuberculous pleurisy, are not specific for tuberculosis. A meta-analysis of 40 articles showed that the sensitivity of ADA testing in pleural fluid for diagnosing tuberculous pleurisy varied widely (from 47% to 100%), much like the specificity (from 50% to 100%) [9]. False-positive results occurred mainly in cases of empyema [8,15 17], rheumatoid pleurisy [15,18], malignancies [8,16], and parapneumonic effusions [16,19]. The sensitivity of IFN-c testing in pleural fluid for diagnosing tuberculous pleurisy was found to range from 57% to 100%, and the specificity from 90% to 100%, according to a meta-analysis of 13 articles [11]. An IFN-c response by activated T-cells can be elicited by many different antigens, and its level can be high in pleural fluids for various reasons, including parapneumonic and neoplastic effusions, especially from haematological malignancies [11 14].

CMI Lee et al. ELISPOT in diagnosing tuberculous pleurisy 177 Pleural effusion of undetermined aetiology: 40 Pleural fluid ELISPOT positive: 21 Pleural fluid ELISPOT negative: 19 FIG. 1. Results of rapid diagnostic tests (pleural biopsy, pleural fluid PCR, sputum PCR, pleural fluid ELISPOT assay) in 19 patients with tuberculous (TB) and 21 patients with non-tb pleural effusions. *MAC, pleurisy due to Mycobacterium avium intracellulare complex. TB: 18 Histology diagnostic: 9 Histology nondiagnostic: 3 Biopsy not done: 6 Fluid PCR: ( ) x 2 Sputum PCR: ( ) x 2 Non-TB:3 B:TB:1 Biopsy: not done Non-TB: 18 Mesothelioma: 1 Cancer: 8 Adeno- Ly mphoma: 1 carcinoma:1 Infarction: 1 MAC*:1 Parapneumonic: 3 Heart failure: 3 MAC*:1 Cytomegalovirus: 1 Thus, a high IFN-c level in pleural fluid is not a finding specific for tuberculous pleurisy. The ELISPOT assay, in contrast, allows detection of spots formed by IFN-c secreted by T-cells that have been stimulated by specific M. tuberculosis antigens. As a technique that allows detection of cells of low abundance that secrete various molecules [31], the assay captured IFN-c immediately after it was secreted, and before it was diluted by the blood or pleural fluid, or degraded, and it allowed the specific detection of as few as ten T-cells per 10 6 PBMCs, a sensitivity much higher than that of conventional ELISA [32]. Thus, it could be more specific and sensitive than the measurement of non-specific ADA or IFN-c in blood or pleural effusion fluid. In the current study, the pleural fluid ELISPOT assay showed high sensitivity in diagnosing tuberculosis, whereas the PBMC ELISPOT assay gave false-negative results in four patients with lymphopenia. The low lymphocyte count probably contributed to the falsenegative results. On the other hand, the reason for the falsenegative results could be that these four patients were immunosuppressed; one was was alcoholic and had been receiving exogenous steroids, one had acute lymphocytic leukemia after marrow transplant, and two were of advanced age (87 years old). The third possible cause of false-negative results could have been that tuberculous pleurisy can be a locally contained disease that has the antigen-specific T-cells compartmentalized in the pleural cavity. Lalvani et al. [23] also reported falsenegative ELISPOT results in lymphopenic patients with pulmonary and/or extrapulmonary tuberculosis. However, the pleural fluid ELISPOT assay was not affected by a low lymphocyte count, as this study showed that antigen-specific, IFN-c-secreting T-cells were concentrated eightto ten-fold in pleural fluid as compared with blood. All four patients with a false-negative blood test were positive according to the pleural fluid ELISPOT assay. False-positive PBMC ELISPOT results were obtained for two patients: one with mesothelioma and the other with heart failure. The cause of the false-positive response was not clear. Given the high incidence of tuberculosis in Taiwan (74.1/10 5 ) [33], the two patients might have been exposed to M. tuberculosis and may have had latent tuberculosis. A false-positive result of the pleural fluid ELISPOT was obtained for three patients, probably due to the nonspecific IFN-c present in the effusion before M. tuberculosis antigen stimulation and trapped at the bottom of the well immediately after the fluid was added. The immunospots in the pleural fluids of these three patients were all smaller than those in true-positive patients, an observation that might be helpful in the interpretation of results in future studies. A definite diagnosis of tuberculous pleurisy depends on the demonstration of M. tuberculosis by acid-fast stain of, or culture from, pleural fluid, sputum or pleural biopsy specimens. The paucity of bacilli in pleural fluid leads to low sensitivity of Ziehl Neelsen staining (0 5%) [7,34 37], as was also observed in this study. Pleural fluid culture has a sensitivity of only 10 37% [4,7,35 37] (42.5% in our study), and requires 2 8 weeks. PCR can be a rapid diagnostic method. However, the reported sensitivity of PCR with pleural fluid varies widely, from 31% to 81% [12,37 40], and in this study it was zero. Thus, for a rapid diagnosis of tuberculous pleurisy, pleural biopsy, followed by histological examination, which has high sensitivity (54 82%) [4,7,35 37], or PCR (90% sensitivity) [37], is often needed. It is, nonetheless, invasive, requires a specialist, and is subject to sampling error. In this study, histological examination of pleural biopsy specimens had a 75% diagnostic yield, but could only be performed in 12 (63%) of the 19 patients, and provided a rapid diagnosis in only nine cases (47%) (Table 3; Fig. 1).

178 Clinical Microbiology and Infection, Volume 15 Number 2, February 2009 CMI The ELISPOT assay was especially useful for patients who could not undergo pleural biopsy, e.g. those with a tendency to bleeding due to hepatic or renal insufficiency, haematological diseases or ongoing anticoagulant therapy, those unable to cooperate due to dementia or mental disorders, those requiring ventilators, or those whose pleural effusions were too small in volume for a safe biopsy. ELISPOT assay results are available overnight, permitting the start of antituberculous chemotherapy that would probably not be started empirically, given the underlying hepatic and/or renal diseases in patients with tendency to bleeding, or multi-organ failure in patients requiring ventilators. 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