The 16-kDa cytosolic antigen of M. tuberculosis was purified to homogeneity by molecular sieving chromatography, and the diagnostic potential of the antigen was evaluated in various categories of patients by enzyme-linked immunosorbent assay (ELISA). The immunoglobulin G (IgG), IgA, and IgM antibody levels to 16-kDa antigen were estimated in the two polar groups, namely, smear-and culture-positive pulmonary tuberculosis (S ؉ C ؉ ) patients and healthy subjects (HS). Sensitivities of 62, 52 and 11% with specificities of 100, 97, and 95% were obtained for the three isotypes, respectively. The total number of positives by a combination of the three isotypes was analyzed in the polar groups, and the sensitivity improved to 83% with a specificity of 93%. Even when a combination of IgG and IgA alone was considered, the sensitivity was 82% with a specificity of 97%. Polyethylene glycol precipitation of the circulating immune complex (CIC) in sera was carried out. The CIC-bound antibodies to 16-kDa antigen were assessed by ELISA in the S ؉ C ؉ , S ؊ C ؉ , and S ؊ C ؊ categories of patients. Measuring the IgG-IgA-IgM combination positivities of the CIC-bound antibodies gave sensitivities of 97.5, 100, and 45.3%, respectively. The specificity of the assay with these combinations was maintained at 95.4%.Tuberculosis is a major health problem throughout the world, resulting in about 3 million deaths annually (17). This contagious disease, though preventable, has had an increased incidence in recent years, mainly due to its association with human immunodeficiency virus disease (30) and also due to the occurrence of multidrug resistance (36). In spite of the availability of an adequate treatment regimen, attempts to restrict and eradicate the disease have failed.The alarming increase in morbidity and mortality due to tuberculosis indicates the need to strengthen control measures. Control of the disease depends largely on early detection and treatment of active cases. There is promise in serodiagnostic tests like enzyme-linked immunosorbent assay (ELISA) because of their ease of performance and cost-effectiveness. Serodiagnosis was attempted in earlier days, using crude and semipurified antigens, and was found to give different ranges of sensitivity and specificity (10,14). The results underline the importance of identifying species-specific antigens and using them for diagnosis.Daniel and Anderson (12) were the first to report on the species-specific antigen 5 of Mycobacterium tuberculosis, which was later proved to be the same as the 38-kDa antigen. As antigen 5, it has shown high specificity in all studies conducted in different parts of the world (88 to 98%), while the sensitivity varied from 49 to 89% (2, 3, 13, 15, 23). Other than this and a limited number of other antigens, species specificity (at least M. tuberculosis complex specificity) is quite rare among the M. tuberculosis antigens.In a preliminary study of immunoblots with patients' sera, we observed the specific recognition of a 16-or 17-kDa band by pooled tuber...

Mycobacterium tuberculosis survives and multiplies inside macrophages of its host by modulating the expression of several genes essential for in vivo survival. An in vivo expression system has been developed, based on green fluorescent protein and kanamycin resistance, to identify M. tuberculosis genes which appear to be up-regulated in infected macrophages. A promoter-trap shuttle vector, pLL192, was constructed, containing a streptomycin resistance gene as selection marker and an artificial bicistronic operon composed of the promoterless green fluorescent protein (gfp) gene, followed by the kanamycin resistance gene. A unique BamHI site upstream of the gfp gene allowed for insertion of promoter libraries. The vector was validated by the use of known regulated or constitutive M. tuberculosis promoters. In addition, an M. tuberculosis genomic DNA library was inserted into pLL192 and then introduced into Mycobacterium bovis BCG. The recombinant BCG cells were then used to infect the J774A.1 murine macrophage-like cell line in the presence of kanamycin. Several recombinant BCG cells were thereby selected that were resistant to kanamycin within infected macrophages, but were sensitive to kanamycin when grown in vitro. The kanamycin resistance phenotype was paralleled by the fluorescence phenotype. After nucleotide sequencing, the corresponding genes were identified as mce1A, PE_PGRS63(RV3097c), Rv2232, Rv1026, Rv1635c, viuB, Rv2231(cobC) and Rv0997. Real-time PCR analysis using RNA isolated at various time points from M. tuberculosis and M. bovis BCG grown in vitro and within macrophages, confirmed the up-regulation of these genes. The level of up-regulation varied from 2-to 40-fold in macrophages compared to growth in vitro.

There is a need for rapid diagnostic methods to identify tuberculosis among human immunodeficiency virus-positive cases (HIV-TB). This study evaluated the serodiagnostic potential of the native 38 kDa and recombinant 27 kDa (mpt 51) antigens of Mycobacterium tuberculosis purified in the laboratory, when applied to HIV-TB patients. The antibody response was studied using enzyme-linked immunosorbent assays (ELISA). In the HIV-TB group, anti-38 kDa antibody of the immunoglobulin G (IgG), IgA and IgM isotypes was found in 38%, 43% and 7%, of patients, respectively. Antibodies to the 27 kDa antigen occurred in 50%, 31%, and 1%, for IgG, IgA and IgM, respectively. The sensitivity increased upon combination of the results of IgG and IgA isotypes for each of the antigens, without compromising specificity. When the results were analysed based on the smear positivity, 71-78% and 54-69% were positive among smear-positive and smear-negative HIV-TB cases, respectively. A higher sensitivity (71% and 69%) was obtained using the 27 kDa antigen. The use of both antigens offered a sensitivity of 82% in smear-positive and 69% in smear-negative cases. There was no difference in antibody response among the HIV-TB cases, related to CD4 counts. Thus, the combination of the 38 and 27 kDa (mpt 51) antigens proved to be of diagnostic utility in HIV-TB, irrespective of the severity of immunosuppression, in smear-positive and smear-negative TB.

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