The diagnosis of pleural tuberculosis (pTB) is difficult, and more sensitive and specific techniques are needed. In the period August 1998 to November 2002, we evaluated 132 patients with a pleural effusion submitted to a thoracentesis and pleural biopsy in a tertiary care hospital in Rio de Janeiro, Brazil. Three tests were performed and compared in the pleural fluid: ADA activity measurement, IgA-ELISA for two combined specific Mycobacterium tuberculosis antigens, and polymerase chain reaction (PCR) for detection of M. tuberculosis DNA. Ninety-five patients (72%) were given a final diagnosis of pTB. Overall histopathologic sensitivity was 77%. The sensitivities of pleural fluid culture and AFB smear were 42% and 1%, respectively. Twenty-one (22%) additional patients had a clinical diagnosis of pTB. Median follow-up time of all TB patients after the completion of antituberculous treatment was 13 months. Sensitivities of ADA, IgA-ELISA and PCR were 91%, 78% and 82%, while specificities were 93%, 96% and 85%, respectively. Only ADA sensitivity was significantly higher than the histopathologic examination (McNemar chi2 test; p = 0.002) and also significantly higher than ELISA (p = 0.049), but not higher than PCR (p = 0.143). We conclude that the routine use of ADA activity measurement in pleural fluid can obviate the need for a pleural biopsy in the initial diagnostic approach to pleural effusions, while IgA-ELISA and PCR techniques, potentially more specific tests, need further refinement to improve their accuracy.

The PstS1 antigen is highly immunogenic, principally when combined with CFP10 during both latent and active TB infection. In the present study, a selected pstS1 gene fragment was cloned, fused with CFP10, and expressed in Escherichia coli. The product M ycobacterium tuberculosis, a highly successful parasite, is the cause of tuberculosis (TB). A major health care concern, the disease results in approximately 1.4 million annual deaths (990,000 among HIV-negative individuals), infecting roughly one-third of the world's population. Among the infected, 5% to 10% will develop active disease in their lifetimes and 90% will harbor the latent form. According to a recent mathematical projection of TB eradication, the treatment of latent TB infection (LTBI) and active TB is urgently required in order to lower and ultimately prevent the further spread of the disease at its present rate (1, 2).Two principal approaches based on the adaptive immune responses elicited by M. tuberculosis infection are currently used to identify TBI (3): the in vivo tuberculin skin test (TST) and the ex vivo gamma interferon (IFN-␥) release assay (IGRA).Developed in 1908, TST became the standard means of assessing the presence of TB infection. Via TST, prior TB exposure is measured by a type 4 delayed-type hypersensitivity reaction when a purified protein derivative (PPD) of M. tuberculosis is injected intradermally. Although TST has some biological limitations such as the occurrence of anergies in one-third of active TB cases, crossreactivity with M. bovis bacillus Calmette-Guérin (BCG) and non-TB mycobacteria does not, according to some authors, result in major sensibility differences in IGRAs (4).The IGRA was recently developed using antigens that are expressed in M. tuberculosis but not in BCG or M. bovis strains as stimuli. These antigens measure the production of IFN-␥ in peripheral blood mononuclear cells (3,5). According to a recent World Health Organization (WHO) report (1), the two currently commercially available IGRAs have yet to generate sufficient data or enough high-quality evidence regarding their performance in the low-and middle-income countries that typically have a high TB and/or HIV burden and where the coverage of BCG vaccination may cause some interference (5, 6).Both IGRAs are based on Mycobacterium tuberculosis-specific antigens, namely, early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP10) (5). These antigens must be validated in different scenarios, but other antigen combinations also need to be evaluated to improve IGRA coverage.The 38-kDa M. tuberculosis antigen, also known as phosphatespecific transporter-1 (PstS-1), coded by a pstS1 gene that composes one of the 3 putative pst operons, is a lipoprotein phosphate transport receptor on the cell surface. These operons probably constitute a subtle biochemical adaptation of M. tuberculosis, enabling it to grow and survive under different phosphate-limiting conditions during its infectious cycle. The PstS-1 protein is ac-

To evaluate commercial Lionex TB together with four antigens of Mycobacterium tuberculosis MT10.3, 16 kDa and 38 kDa)

The objective of this study was to evaluate people attending a primary health clinic in Rio de Janeiro, Brazil for immunoreactivity to five Mycobacterium tuberculosis antigens, as these antigens are markers of immune response and factors associated with active TB. The serum antibody titers of different categories of patients (defined by microbiological and radiological characteristics and by response to therapy on follow-up) to 38 kDa, 16 kDa, MPT64, ESAT-6 and MT10.3 antigens were determined blind with ELISA. Positive tests to each antigen were defined with ROC analysis. OR were calculated for factors associated with humoral response in patients with active TB. A total of 201 patients underwent serological testing. Patients with confirmed active TB responded more frequently to MPT64 (44%), 16 kDa (37.7%) and 38 kDa (36.1%). ESAT-6 and MT10.3 were also able to distinguish people in TB groups from controls. TB infected subjects responded less frequently to ESAT-6 and MT10.3 (3.7% and 11%, respectively). Sensitivity and specificity to all antigens combined were 58.4% and 60.7%, respectively. Reactivity to 38 kDa and to MPT64 was more likely among alcohol users OR 2.61 (95%CI;1.05-6.94) and OR 3.27 (95%CI;1.33-8.15), respectively. 16 kDa antigen elicited a more protective response among smokers, OR 0.29 (95%CI; 0.10-0.83). It was concluded that reactivity to all antigens tested represented markers of active disease. ESAT-6 and MT10.3 could not be identified as markers of TB infection in this community. Sensitivity was higher to all antigens combined, but at a cost of lower specificity. Interestingly, among factors associated with positive immunoreactivity, alcohol use and smoking seem to polarize the humoral response in different directions. This finding deserves further investigation.

Background A previous study demonstrated pleural fluid (PF) IgA immunodominance for the fused MT10.3:MPT64 protein in pleural tuberculosis (PLTB) cases. However, no clue on the role of IgA and IgG against this and other antigens in PF and serum concerning improved diagnosis is available. Thus, the aim of the present study was to validate PF IgA-MT10.3:MPT64 and evaluate PF and serum IgA and IgG reactivity against this protein, its peptides (F2) and single MPT64, MT10.3 and the PPE59 mycobacterial specific antigens. IgA and IgG ELISA were measured against the antigen in PLTB (n = 29) and other non-TB pleurisy (n = 39) patient samples. Results The immunodominance of PF IgA-MT10.3:MPT64 was confirmed in PLTB (86.2%) followed by PPE59 (62%), while serum IgA-F2 exhibited 51.7% sensitivity. PF and serum IgG-MT10.3:MPT64 led to 65.5 and 51.7% sensitivity, respectively. However, MT10.3 and MPT64 displayed overall lower sensitivity (≤34.5) for both antibodies. All results at 95% fixed specificity. Combinatory results indicated 93.1% sensitivity for PF IgA-MT10.3:MPT64/−PPE59 and IgA/IgG-MT10.3:MPT64 at 92.3% specificity, followed by IgA-MT10.3:MPT64/−MPT64 or /−F2 (89.6%) without jeopardizing specificity (94.9%). The combinatory results of the PF adenosine deaminase test (ADA) and IgA-MT10.3:MPT64/−F2 demonstrated the highest sensitivity (96.6%), with a specificity of 92.3%. Conclusions The PF IgA-MT10:MPT64 immune dominance was validated in PLTB, and its combinatory results with PPE59 or MPT64 or F2 antigens as well as with IgG, are reported herein for the first time, improving their potential to assist diagnosis. Combining PF-ADA and IgA-MT10.3:MPT64/−F2 results achieved better accuracy. Moreover, serum IgG, although less accurate, displays potential beyond microbiological tests.

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Background: The role of protein families PE/PPE remains relatively enlightening. The gene rv3429, coding the PPE59 protein, is one of the 12 members of the PPE subfamily, and it is absent in all BCG strains. Although elicits low T cell response, there are no clues for the ability to induce a humoral response. Methods: We cloned, expressed, and analyzed by ELISA IgG, IgM and IgA anti PPE59 in 212 sera. Of them, 69 were from tuberculosis patients’ residents in Italy (TBIT, 12 native and 67 immigrants), the remaining 133 are Brazilians citizens (BR). Pulmonary (pTBIT) or extrapulmonary (eTBIT) clinical forms were 54 or 25. The pTBBR were 52 and 81 non-TB patients, including 10 non-tuberculous mycobacteria infected (NTM). Results: Keeping the specificity at 97%, IgA sensitivity decreased from pTBBR (53%) to pTBIT (38%) and eTBIT (28%), with an overall sensitivity of 42.7% and moderate accuracy (61.8 %), while IgG showed significant lower (p<0.0001) performance, 21%, 3%, 0%, 9% and 37%, respectively, at specificity of 83%. Groups were not discriminate by IgM. False-positive NTM IgG was high. Combination 16kDa IgG, previously performed only on Bazilians’ sera, plus PPE 59 IgA results increased sensitivity to 71% at 87.4% specificity. The overall smear microscopy (SM) diagnosis was 70.8% and a combination of SM/IgA increased sensitivity to 74% (p=0.01), mainly among SM- cases. Among pTBBR, all these rapid tests, including SM, sensitivity reach 86.5% (p=0.001). Positive IgA polarization was significant in extensive lung disease (p=0.001) and alcohol users (p=0.04). Conclusion: Although PPE59 IgA independently has moderate accuracy on TB diagnosis, together with other biomarkers contributes to improving its detection. Moreover, the polarized reactivity deserves further investigation.

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