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Universidade Federal do Rio de Janeiro, Rio de Janeiro, † Fundação Técnico-Educacional Souza Marques, Rio de Janeiro,. ‡ Laboratório de Hanseníase ...
INT J TUBERC LUNG DIS 9(4):461–466 © 2005 The Union

IgA antibody responses to Mycobacterium tuberculosis recombinant MPT-64 and MT-10.3 (Rv3019c) antigens in pleural fluid of patients with tuberculous pleurisy M. C. Kaisermann,*† I. G. Sardella,‡ A. Trajman,†§ L. V. Coelho,§ S. Kämpfer,¶ F. Jonas,¶ M. Singh,¶ M. H. F. Saad‡ * Universidade Federal do Rio de Janeiro, Rio de Janeiro, † Fundação Técnico-Educacional Souza Marques, Rio de Janeiro, ‡ Laboratório de Hanseníase, Fundação Oswaldo Cruz, Rio de Janeiro, § Universidade Gama Filho, Rio de Janeiro, Brazil; ¶ Lionex Diagnostics & Therapeutics GmbH, Braunschweig, Germany SUMMARY S E T T I N G : Microbiological tests lack sensitivity for pleural tuberculosis (TB) and histopathology is expensive, time consuming and needs specialised personnel. Immunoassay (ELISA) may be a promising approach in this respect. O B J E C T I V E : To evaluate the reactivity of IgA antibody to MPT-64 and MT-10.3 recombinant mycobacterial protein antigens in pleural fluid as a marker of pleural TB, based on the fact that IgA is the main antibody in the mucosa/serosa of the gastrointestinal and respiratory tract. M E T H O D : Anti-MPT-64 and MT-10.3 IgA response was determined by ELISA in 72 patients with pleural TB and 27 with other pleural conditions. R E S U L T S : High sensitivities for IgA were measured

against MPT-64 (52/72, 72%) and MT-10.3 (52/72, 72%) antigens. Combining the sensitivities of both antigens, further increase in sensitivity (55/72, 76%) was obtained with no loss of specificity (96%). Similar IgA reactivity was obtained from culture-negative and culture-positive specimens. In eight pleural TB patients with human immunodeficiency virus (HIV) co-infection, the sensitivity was 88% (7/8). C O N C L U S I O N : To our knowledge, this is the first description of the presence of IgA antibody pleural TB effusion reactive to MPT-64 and MT-10.3, with sensitivity similar to histopathological examination, which is presently considered the gold standard for pleural TB. K E Y W O R D S : pleural tuberculosis; IgA; recombinant antigen; MPT-64; MT-10.3

THE DIAGNOSIS of tuberculosis (TB) is still a major challenge. Acid-fast bacilli (AFB) identification in tissue or body fluids is currently the fastest and cheapest way to diagnose the disease, but up to 40–60% of patients with pulmonary disease and up to 90% of those with extra-pulmonary TB (EPTB) may be smearnegative.1,2 Culture is the gold standard for the diagnosis of TB, but although highly specific, its sensitivity varies from 60% to 80% in sputum to less than 20–40% in pleural fluid.2,3 Moreover, the final results of culture may take 4 to 6 weeks. The pleura is one of the most frequent sites of EPTB,1,2 and demonstration of granuloma in pleural specimens is currently considered the gold standard for the diagnosis of pleural TB. However, pleural biopsy increases morbidity of thoracentesis, needs trained personnel and an adequate setting and leads to increased health costs. In the last decades a number of new diagnostic

techniques have been tested. However, none has shown sufficient sensitivity, specificity or affordability to replace conventional tests in low-income countries, where 90% of new cases occur.1,2 Identification of Mycobacterium tuberculosis DNA by polymerase chain reaction (PCR) in respiratory specimens is only accepted for the diagnosis of pulmonary TB.4 For EPTB diagnosis, PCR is a controversial method because it lacks reproducibility, as seen in several reports of large variations in sensitivity and specificity.5–7 Humoral response is considered non-protective against intracellular pathogens. Nevertheless, antibodies can protect against many viruses, all of which are obligate intracellular pathogens,8 or even against more complex intracellular microorganisms such as Toxoplasma gondii.9 Although the role of antibodymediated immunity in protection against M. tuberculosis remains uncertain, exposure to M. tuberculosis does elicit the production of antibodies to several

Correspondence to: Dr Maria Helena Féres Saad, Laboratório de Hanseníase-IOC, Fundação Oswaldo Cruz, Av. Brasil 4365, Rio de Janeiro, CEP 21045-900, Brazil. Tel: (155) 21 2598 4288. Fax: (155) 21 2270 9997. e-mail: saad@ioc. fiocruz.br Article submitted 11 May 2004. Final version accepted 13 August 2004.

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antigens, which could be used as markers of TB infection.10–14 Among the five most common classes of immunoglobulins, IgG and IgM are usually explored in studies on TB diagnosis through the detection of these antibodies in serum and other body fluids using different single antigens. Measuring IgA, the antibody found in the mucosa of the gastro-intestinal and respiratory tracts,15 has not been well evaluated in diseases such as pleural TB. The purpose of the present study was to evaluate IgA reactivity to two recombinant protein antigens, MPT-64 and MT-10.3, in the pleural effusion of patients with pleural TB and other pleural diseases, to assess their diagnostic potential.

MATERIALS AND METHODS Patients and clinical setting Ninety-nine patients with pleural effusion consecutively admitted for diagnosis between January 1999 and July 2001 at the Hospital Geral da Santa Casa de Misericórdia, a tertiary care general hospital in Rio de Janeiro city, Brazil, were included. Brazil is an endemic area for TB, and Rio de Janeiro city has an incidence rate of 170 per 100 000 population. The inclusion criterion was the presence of pleural effusion with clinical indication for thoracentesis. Patients not willing to participate or give informed consent and those on an anti-tuberculosis regimen for more than 7 days were excluded. Patients were classified into three groups, by diagnosis: Group I comprised patients with proven diagnosis of pleural TB by positive AFB staining in pleural fluid or sputum, or a positive culture in sputum, pleural fluid or tissue, and by the finding of granuloma in pleural tissue. Group II comprised patients whose pleural TB diagnosis was based on clinical findings, defined as fever, night sweats for .15 days, a lymphocytic exudative pleural effusion with a non-diagnostic pleural biopsy (with positive or negative culture), and a good response to specific treatment. Patients with other pleural conditions constituted Group III (nonpleural TB). None of the patients had purified protein derivative (PPD) skin test or bacille Calmette-Guérin (BCG) vaccination information available. Patients underwent blood and pleural fluid tests for lactic dehydrogenase (LDH), glucose, cholesterol, amylase, protein and albumin concentration and total and differential cell count, AFB and Gram staining (data not shown). Human immunodeficiency virus (HIV) infection was tested using ELISA (Genscreen HIV1/2, BioRad, Hercules, CA, USA). A sample of spontaneous or induced sputum was obtained for AFB staining and culture in Löwenstein-Jensen medium (LJ). Four fragments of parietal pleura were obtained with a Cope needle, three for histopathological examination and one for LJ culture. Pleural fluid was aliquoted in microcentrifuge tubes and stored at 2708C until use.

Pleural fluid was defrosted and centrifuged (Eppendorf, Sigma, St Louis, MO, USA) at 14.000 rpm for 10 min, and the immune assay was performed on the supernatant. Specific M. tuberculosis recombinant antigens MPT64 and MT-10.3 (Rv3019c) were prepared as described previously.16,17 Antigens were diluted at a concentration of 1 mg/ml in phosphate buffered saline (pH 5 7.4). Fifty microliters of each of the antigens were placed in different ELISA microplate wells (Nunc, flat bottomed, immunoplate C96 Maxisorp, Thomas Sci, Swedesboro, NJ, USA) and incubated for 2 h at 378C. Non-specific binding was blocked by adding to each well 100 ml 5% bovine serum albumin (BSA, fraction V, Sigma) in phosphate buffered saline containing 0.01% Tween 20 (PBSt) and incubated as above. The plates were then washed four times with 200 ml PBSt and used immediately or stored at 48C in a sealed plastic bag for no more than 1 week. For protein multi-antigen enzyme-immunoassay (Prot-EIA), testing of pleural fluid supernatants was performed with samples diluted to 1:100 with 1% BSA-PBSt, and 50 ml of each sample was placed in the antigen-treated microplate wells. After incubation at 378C for 1 h, the plates were washed as described above and 50 ml of goat anti-human IgA peroxidase conjugates (Pierce, Rockford, IL, USA), previously diluted to 1:5000 in 1% BSA-PBSt, was added to each well and incubated and washed as above. The reactions were developed by adding 50 ml of TMB (trimethylbenzidine solution, SH, (Pierce) and incubated at room temperature for 15 min in a dark room. The reaction was stopped by the addition of 100 ml of a 2.5M H2SO4 solution. The intensity of the reactions was read using a Labsystems Multiskan MS Plate Reader (Stockholm, Sweden) at two wavelengths (450/630 nm) with continuous movement, to control for variations of absorbance (condensation, fingerprint). All tests were done in duplicate and each sample was tested three times on different days. In each set of experiments, positive (pooled TB) and negative (pooled control) pleural fluid specimens were used as references. The positive and negative pools consisted of a mixture of 50 ml of 10 samples of TB pleural fluid from patients with a positive pleural biopsy and six samples of non-pleural TB pleural fluid from patients diagnosed by histopathological examination. Statistical analysis To calculate the potential diagnostic value of IgA in pleural effusion, a cut-off point was established, which is generally defined as the average of a control population (Group III) plus two standard deviations. Results are expressed as optical density (OD) 3 1000. Tests were considered positive or negative when consistent results were obtained in three repeated tests. For comparison of mean titers, an analysis of variance was performed, then Student’s t-test was used to

IgA in tuberculosis pleural fluids

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Figure Individual MPT-64 and MT-10.3 IgA response in 72 pleural tuberculous patients (PTB, Group I 5 52 and Group II 5 20) and in 27 non-tuberculous patients (non-PTB). OD 5 325 and 371 (long bars) are the cut-off of the MPT-64 and the MT-10.3 IgA-Prot-EIA test, respectively. Short bars 5 IgA mean of each patient group. OD 5 optical density.

compare means among groups. K-test was used to analyse the immune response homogeneity to the antigens. Data processing was performed using SPSS 9.0.1 (SPSS, Chicago, IL, USA) and Prism3 (GraphPad Software Inc, San Diego, CA, USA).

RESULTS Of 99 analysed pleural effusion samples, 72 were from patients with pleural TB: 52 comprised Group I and 20 comprised Group II. Group III patients had the following diagnoses: lung cancer (10), parapneumonic effusion (5), heart failure (4), renal failure (2), lymphoma (1) and systemic lupus erythematosus (1). In four additional patients no diagnosis was obtained despite thorough investigation. Although their clinical findings were not consistent with pleural TB, two of them received anti-tuberculosis drugs with no clinical improvement. Eight of the 72 (11%) pleural TB and one of the 27 (4%) Group III patients were HIVpositive. Only one pleural TB patient elicited an AFBpositive specimen. Positive cultures were obtained from 10 pleural biopsies (eight from Group I and two from Group II, 13.8%). No significant difference was found in the mean age of the pleural TB (40 6 18) and non-pleural TB patients (58 6 12, P 5 0.06), although the latter group was slightly older. The Figure shows anti-MPT-64 and anti-MT-10.3 IgA measurements and mean values in pleural fluid specimens in the different groups. Cut-off values in OD established in Group III were defined as respectively 325 and 371. The mean OD for IgA was 404 6 247 and 466 6 260 for pleural TB specimens and 173 6 79 and 173 6 79 for Group III specimens (P , 0.0001) for MPT-64 and MT-10.3 antigens, respectively. Among the eight HIV-positive patients with pleural TB, the mean OD was similar to those in the HIV-

negative patients (473 6 245/490 6 277 and 457 6 270/535 6 277, P 5 0.11, for MPT-64 and MT-10.3, respectively). The mean OD in 10 pleural fluid specimens from patients with M. tuberculosis positive culture was similar to those with a negative culture (435 6 115/487 6 110, P 5 0.4 and 466 6 268/522 6 274, P 5 0.3 for MPT-64 and MT-10.3, respectively). Sensitivity, specificity, positive and negative predictive values of Prot-EIA for IgA results with the recombinant antigens are shown in the Table. Anti-MPT-64 and anti-MT-10.3 IgA reacted in the same way in both groups of pleural TB (k 5 0.80), thus IgA against both antigens was detected in 72% of pleural TB specimens, contrasting with 4% in Group III. The patient with a false-positive test had lung cancer and did not have a known history of previous TB. The cumulative results for both antigens increased the sensitivity to 76% with no change in the specificity, given that two groups of three different patients recognised only one of the antigens. Among the eight HIV-positive patients with pleural TB, anti-MPT-64 and antiMT-10.3 IgA were detected in 63% (5/8) and 88% (7/ 8) of the patients, respectively. Fourteen of 20 pleural TB patients with a non-specific pleural biopsy (Group II), two of whom had a positive culture, showed an IgA immune response for both antigens; thus IgA immuno-assay detected 70% of TB patients missed by histopathological examination.

DISCUSSION Among extra-pulmonary presentations of TB, pleurisy has the second highest frequency.1,18 Conventional methods for the diagnosis of pleural TB lack sensitivity, are expensive and results are often delayed.19 The best available method to diagnose pleural TB is histopathological examination of pleural

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Table Sensitivity, specificity, PPV and NPV of the Prot-EIA test for IgA pleural fluid using the recombinant MPT-64 and MT-10.3 Mycobacterium tuberculosis antigens Sensitivity* Antigens MPT-64 MT-10.3 MPT-641MT-10.3

IgA

Group I‡ n (%)

Group II‡ n (%)

Overall‡ n (%)

Specificity† n (%)

PPV %

NPV %

38/52 (73) 38/52 (73) 41/52 (79)

14/20 (70) 14/20 (70) 14/20 (70)

52/72 (72) 52/72 (72) 55/72 (76)

26/27 (96) 26/27 (96) 26/27 (96)

72 72 76

96 96 96

* Sensitivity 5 number of positive ELISA TB pleural fluid/total of TB pleural fluid 3 100. † Specificity 5 number of negative non-TB pleural fluid/total of non-TB pleural fluid 3 100. ‡ Group I: patients with proven diagnosis of pleural TB by positive AFB in pleural fluid or sputum; or a positive culture in sputum, pleural fluid or tissue and granuloma in pleural tissue. Group II: patients whose pleural TB diagnosis was based on clinical findings such as fever, nights sweats .15 days, a lymphocytic exudative pleural effusion with a non-diagnostic pleural biopsy and a good response to specific treatment. PPV 5 positive predictive values; NPV 5 negative predictive values; TB 5 tuberculosis; AFB 5 acid-fast bacilli.

tissue, which has a reported 80% sensitivity in the best series. However, pleural biopsy increases the morbidity of thoracentesis, especially in children,20 and needs specialised personnel to process and interpret the specimen, which increases the costs of diagnosis. In our study, the sensitivity of culture was 1.4% and the sensitivity of histopathological examination was 74%. Therefore, in many situations, pleural TB is diagnosed exclusively on clinical grounds and is often empirically treated, a practice not devoid of risks, such as drug toxicity.21 The detection of IgA in 72% of pleural TB specimens in this study, a rate close to that of histopathological examination, suggests that an ELISA-based test for IgA may be useful for confirming suspected tuberculous pleurisy. The performance of the test seems to be unaffected in HIV-infected patients or culture-negative specimens. Although the number of HIV-positive patients was low among our subjects, our findings confirm previous reports that immunological pleural effusion tests may not be altered by HIV serological status.22 This may also be explained by the fact that all HIV-infected patients presented pleural TB as the first manifestation of HIV infection, suggesting the first stages of immune suppression. In our samples, the optical density did not differ among culture-positive and -negative specimens, suggesting that bacillary load may not be directly related to the production of IgA. The IgA reactivity among pleural TB patients showed significant homogeneity against both tested antigens, in agreement with previous reports showing that TB patients elicit homogeneous reactivity against well-defined subsets of antigens and that the use of only one antigen allowed recognition of antibodies in 70% of TB patients with or without HIV co-infection.23,24 To improve the sensitivity of antibody detection, additional antigens may be helpful, as seen in this study, where a combination of MPT-64 and MT-10.3 slightly increased the sensitivity without changes in the specificity. To our knowledge this is the first study to show the IgA host pleural response to MPT-64 and MT-10.3 recombinant antigens. IgM and IgG response to these antigens was weak (data not shown), with low sensi-

tivity, perhaps due to the fact that IgM is produced early in the course of the disease.15 As pleural TB onset is insidious, when patients seek medical care a switch to other immunoglobulin classes may already have occurred. Low IgG reactivity may be explained by a weak systemic immunological response in contrast to a strong local one, assuming that intrapleural IgA production occurred during the inflammatory reaction. Other authors have measured IgG and IgM levels in TB patients’ pleural fluid and serum. Kunter et al. reported that pleural fluid anti-A60 IgM had the highest sensitivity (77%) and specificity (94%);25 however, the NPV was very low (40%), contrasting with our IgA results which showed similar sensitivity (76%) and specificity (96%), but a higher NPV (96%). This may suggest that MPT-64 and MT-10.3 antigens are more specific than A60. Levy et al. found no difference between pleural fluid and serum antibody level and concluded that the presence of IgG in pleural fluid was due to passive diffusion.26 Conversely, others have concluded that cellular immune response in pleural TB is compartmentalised in the pleural space, as pleural T-cells showed a marked proliferative response to PPD while peripheral blood T-cells were not reactive.21,27 In the same way IgA, the predominant immunoglobulin in the mucosa/serosa environment15 is produced in the pleural space.28 Unfortunately we did not have patients’ serum available to compare the results. However, IgA has been found against P-90 antigen in the serum of pulmonary TB patients.24 Prot-EIA tests are inexpensive, easy to perform, do not need specialised personnel and can be implemented in virtually any reference laboratory and hospital. The test seems to have some limitations, however; falsepositive ELISA results in patients with lung cancer have been reported by other authors and may be explained by a history of previous healed TB29 or possibly disorders in the immune response elicited in the course of non-tuberculous diseases, considering that most M. tuberculosis antigens may share some similarities with other mycobacterial species, such as BCG and atypical mycobacteria. MPT-64 has been reported to be a highly specific antigen that distinguishes

IgA in tuberculosis pleural fluids

TB infection from sensitisation by BCG, although it is expressed in some strains of BCG.30 MT-10.3 belongs to the esat-6 subfamily and is present also in M. avium.31 In our study, however, only one of 10 lung cancer patients had a false-positive result. Bleeding in the collected sample should be avoided, as haemolysis may interfere with ELISA results.32 Further clinical studies with larger sample sizes are necessary to validate the test in a routine clinical setting. Other antigens should also be assayed to enhance the sensitivity of the test. Acknowledgements Financial support gratefully received from the Cooperation Program Brazil x Germany (International Buero, DLR, BMBF— Sciences and Technology Ministry); Johns Hopkins University with funds provided by grant number 1U19AI45432-01 from the National Institute of Health; CNPq (Projeto Milênio) and FAPERJ grant E-26/170.467/2001. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

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RÉSUMÉ

La sensibilité des tests microbiologiques pour la tuberculose pleurale est faible et l’examen histopathologique est coûteux, de longue durée et nécessite un personnel spécialisé. A cet égard, un test immunologique du type ELISA pourrait être une approche prometteuse. O B J E C T I F : Evaluer la réactivité des anticorps IgA à l’égard des antigènes protéiques mycobactériens recombinants MPT-64 et MT-10.3 dans le liquide pleural comme marqueurs de la pleurésie tuberculeuse en se basant sur le fait que l’IgA est le principal anticorps dans les muqueuses et les séreuses des tractus gastro-intestinal et respiratoire. M É T H O D E : On a déterminé par un test ELISA la réponse IgA anti-MPT-64 et MT-10.3 chez 72 patients atteints de tuberculose pleurale et chez 27 patients atteints d’autres affections pleurales. CONTEXTE :

Les sensibilités des IgA sont élevées contre l’antigène MPT-64 (52/72, 72%) et contre l’antigène MT-10.3 (52/72, 72%). Si l’on combine la sensibilité des deux antigènes, une augmentation de sensibilité est obtenue (55/72, 76%) sans perte de spécificité (96%). La réactivité des IgA a été similaire qu’il s’agisse d’échantillons à culture négative ou positive. La sensibilité reste à 88% (7/8) chez les huit patients atteints de tuberculose pleurale et co-infectés par le virus de l’immunodéficience humaine. C O N C L U S I O N : A notre connaissance, il s’agit de la première description de la présence d’anticorps IgA réagissant à MPT-64 et MT-10.3 dans les épanchements pleuraux tuberculeux ; la sensibilité est similaire à celle de l’examen histopathologique, qui est actuellement considéré comme le « gold standard » pour la tuberculose pleurale. RÉSULTATS :

RESUMEN M A R C O D E R E F E R E N C I A : Las pruebas microbiológicas tienen una sensibilidad deficiente para el diagnóstico de tuberculosis pleural y el estudio histopatológico es costoso y precisa tiempo y personal especializado. En estas condiciones, el análisis inmunoenzimático (ELISA) podría constituir una alternativa promisoria. O B J E T I V O : Analizar la reactividad del anticuerpo de tipo IgA del líquido pleural a los antígenos proteicos micobacterianos producidos biotecnológicamente, MPT64 y MT-10.3, como signo indicador de tuberculosis pleural ; se parte del hecho que la IgA es el principal anticuerpo de las mucosas y serosas de los aparatos gastrointestinal y respiratorio. M É T O D O : Se midió mediante ELISA la respuesta de IgA dirigida contra el MPT-64 y el MT-10.3 en 72 pacientes con tuberculosis pleural y en 27 con otras patologías pleurales.

Se observó una alta sensibilidad de la respuesta IgA dirigida contra los antígenos MPT-64 (52/ 72 ; 72%) y MT-10.3 (52/72 ; 72%). Los resultados acumulados con ambos antígenos incrementan aún más la sensibilidad (55/72 ; 76%), sin pérdida de especificidad (96%). La reactividad de la IgA fue equivalente en muestras con cultivo positivo y cultivo negativo. En ocho pacientes con tuberculosis pleural y coinfección por el virus de la inmunodeficiencia humana la sensibilidad fue del 88% (7/8). C O N C L U S I Ó N : Esta es la primera descripción, en nuestro conocimiento, de la presencia de anticuerpos del tipo IgA en el líquido pleural, que reaccionan contra los antígenos MPT-64 y MT-10.3, con una sensibilidad equivalente a la del estudio histopatológico, considerado actualmente el examen de referencia para diagnóstico de tuberculosis pleural. RESULTADOS :