Virulence‐dependent induction of interleukin‐10‐producing‐tolerogenic dendritic cells by Mycobacterium tuberculosis impedes optimal T helper type 1 proliferation

Mycobacterium tuberculosis inhibits optimal T helper type 1 (Th1) responses during infection. However, the precise mechanisms by which virulent M. tuberculosis limits Th1 responses remain unclear. Here, we infected dendritic cells (DCs) with the virulent M. tuberculosis strain H37Rv or the attenuated strain H37Ra to investigate the phenotypic and functional alterations in DCs and resultant T‐cell responses. H37Rv‐infected DCs suppressed Th1 responses more strongly than H37Ra‐infected DCs. Interestingly, H37Rv, but not H37Ra, impaired DC surface molecule expression (CD80, CD86 and MHC class II) due to prominent interleukin‐10 (IL‐10) production while augmenting the expression of tolerogenic molecules including PD‐L1, CD103, Tim‐3 and indoleamine 2,3‐dioxygenase on DCs in a multiplicity‐of‐infection (MOI) ‐dependent manner. These results indicate that virulent M. tuberculosis drives immature DCs toward a tolerogenic phenotype. Notably, the tolerogenic phenotype of H37Rv‐infected DCs was blocked in DCs generated from IL‐10^−/− mice or DCs treated with an IL‐10‐neutralizing monoclonal antibody, leading to restoration of Th1 polarization. These findings suggest that IL‐10 induces a tolerogenic DC phenotype. Interestingly, p38 mitogen‐activated protein kinase (MAPK) activation predominantly mediates IL‐10 production; hence, H37Rv tends to induce a tolerogenic DC phenotype through expression of tolerogenic molecules in the p38 MAPK–IL‐10 axis. Therefore, suppressing the tolerogenic cascade in DCs is a novel strategy for stimulating optimal protective T‐cell responses against M. tuberculosis infection.     

Relationship Between Tuberculin Hypersensitivity and Cellular Immunity to Infection in Mice Vaccinated with Viable Attenuated Mycobacterial Cells or with Mycobacterial Ribonucleic Acid Preparations

The migration inhibition technique has been used to study delayed hypersensitivity in vitro by using peritoneal exudate cells and splenic lymphocytes from mice vaccinated with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis and from mice vaccinated with ribonucleic acid (myc RNA) preparations obtained from viable mycobacterial cells of the same strain. Inhibition of macrophage migration was noted when purified protein derivative (PPD) or viable H37Ra cells were added to peritoneal exudate cells obtained from mice immunized with viable H37Ra cells and not from mice immunized with myc RNA. Splenic lymphocyte cultures were exposed to the same antigens in vitro. Filtered supernatant fluids from these lymphocyte cultures, when added to peritoneal exudate cells obtained from nonimmunized mice, inhibited migration only when they were obtained from lymphocytes which came from mice immunized with viable H37Ra cells. Injection of PPD intravenously into vaccinated mice resulted in inhibitory supernatant fluids from splenic lymphocyte cultures only when the lymphocytes came from mice immunized with viable H37Ra cells. However, intravenous injection of either viable H37Ra cells or of myc RNA preparations into mice vaccinated with myc RNA occasionally produced inhibitory supernatant fluids when lymphocytes were obtained from these mice. On the other hand, mice vaccinated with myc RNA or viable H37Ra cell preparations were consistently and equally protected against intravenous challenge with the virulent H37Rv strain. Thus, although some evidence was obtained for a delayed type hypersensitivity in mice vaccinated with H37Ra cells or with myc RNA to ribosomal proteins or other proteins associated with the RNA preparation, no evidence of tuberculin hypersensitivity could be detected in any mice vaccinated with the myc RNA. These results argue against a role for tuberculin hypersensitivity in immunity to tuberculous infection.     

Differential Growth Characteristics and Streptomycin Susceptibility of Virulent and Avirulent Mycobacterium tuberculosis Strains in a Novel Fibroblast-Mycobacterium Microcolony Assay

The ability to spread from cell to cell may be an important virulence determinant of Mycobacterium tuberculosis. An in vitro assay was developed to characterize this ability among four strains of M. tuberculosis: the attenuated strain H37Ra, the virulent strains H37Rv and Erdman, and a virulent clinical isolate (Stew). Confluent monolayers of human skin fibroblasts were infected with these strains and overlaid with agar-medium. M. tuberculosis infection developed over 21 days as microcolonies originating within the plane of the fibroblasts. Microcolonies of the virulent strains had an elongated appearance and exhibited extensive cording. The cords appeared to invade adjacent cells within the plane of the monolayer. Microcolony diameter of the Erdman strain was significantly larger than that of the other virulent strains, indicating that virulent strains can have distinguishing phenotypes in this assay. In contrast, avirulent H37Ra microcolonies were rounded and noncorded. H37Ra microcolonies were significantly smaller than those of the virulent strains. Microcolony diameter of the virulent strains was not reduced by the extracellularly acting antibiotic streptomycin at concentrations of up to 5.0 μg/ml. In contrast, H37Ra microcolony size was reduced at concentrations as low as 0.5 μg/ml. Growth of all strains was similarly inhibited by 1.0 μg of streptomycin per ml in fibroblast-conditioned tissue culture medium alone. When fibroblasts were infected with the M. tuberculosis strains without an agar overlay, with and without streptomycin, numbers of CFU mirrored the changes observed in the microcolony assay. There was a statistically significant decrease in H37Ra CFU compared to virulent strains after treatment with streptomycin. These differences between H37Ra and virulent strains in human fibroblasts suggest that H37Ra may be lacking a virulence determinant involved in cell-to-cell spread of M. tuberculosis.     

Isolation of two antigens from the culture filtrates of Mycobacterium tuberculosis and their applications in the laboratory diagnosis of the tuberculous meningitis

Two antigens were isolated from the culture filtrates of H₃₇Ra Mycobacterium tuberculosis by immunoabsorbent affinity chromatography, M. tuberculosis antigen 5 and immunoabsorbent affinity column-purified antigen (IAP). The potential application of these two mycobacterial antigens in the laboratory diagnosis of tuberculous meningitis was evaluated by indirect enzyme-linked immunoabsorbent assay in cerebrospinal fluid specimens. IAP antigen was more sensitive than antigen 5, although antigen 5 was more specific than IAP antigen in detecting tuberculous aetiology. Technical aspects of immunoabsorbent affinity chromatography have been highlighted in this study.     

Expression of Virulence of Mycobacterium tuberculosis within Human Monocytes: Virulence Correlates with Intracellular Growth and Induction of Tumor Necrosis Factor Alpha but Not with Evasion of Lymphocyte-Dependent Monocyte Effector Functions

We assessed the applicability of an in vitro model of low-level infection of human monocytes to the characterization of the virulence of strains of the Mycobacterium tuberculosis family. Peripheral blood monocytes were infected at a 1:1 ratio with the virulent M. tuberculosis strain H37Rv, the avirulent M. tuberculosis strain H37Ra, and the attenuated M. bovis strain BCG. Both the percentages of cells infected by the three strains and the initial numbers of intracellular organisms were equivalent, as were levels of monocyte viability up to 7 days following infection. Intracellular growth reflected virulence, as H37Rv replicated in logarithmic fashion throughout the assay, BCG growth reached a plateau at 4 days, and H37Ra did not grow at all. The same patterns of growth were observed following infection of human alveolar macrophages with H37Rv and H37Ra. Monocyte production of tumor necrosis factor alpha was significantly higher following infection with virulent H37Rv than with either BCG or H37Ra. In contrast, there was no clear correlation of interleukin 10 production with virulence. Nonadherent cells of purified-protein-derivative-positive donors mediated equivalent degrees of reduction of the intracellular growth of H37Rv, BCG, and H37Ra. Low-level infection of human monocytes with H37Rv, BCG, and H37Ra thus provides an in vitro model for assessment of the virulence of these M. tuberculosis family strains. Furthermore, it is suggested that the virulence of these strains is expressed primarily by their differing abilities to adapt to the intracellular environment of the mononuclear phagocyte.     

Improved sensitivity of diagnosis of tuberculosis in patients in Korea via a cocktail enzyme-linked immunosorbent assay containing the abundantly expressed antigens of the K strain of Mycobacterium tuberculosis

Tuberculosis (TB) is the leading cause of death from a single infectious agent in Korea. In this study, we compared the proteins present in culture filtrates from Mycobacterium tuberculosis strain K, which is the dominant clinical isolate in Korea, with those present in culture filtrates from M. tuberculosis H37Rv. Several differences in expression were detected between the two strains for those proteins with a molecular mass of <20 kDa. ESAT-6, HSP-X, and CFP-10 were found to be abundantly expressed in the strain K culture filtrates by liquid chromatography-electrospray ionization-time of flight mass spectrometry. The serodiagnostic potentials of recombinant antigens rESAT-6, rHSP-X, and rCFP-10 and two native antigens (Ag85 and PstS1) were evaluated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera collected from 46 TB patients with active disease and 46 healthy controls. As for our ELISA results, HSP-X was superior to the other antigens in terms of sensitivity when a single antigen was employed. The results of a receiver operator characteristic analysis revealed that a cocktail ELISA using all five antigens was significantly more sensitive (77.8%) than the use of a single antigen and offered equivalent specificity; moreover, it produced the largest area under the curve (0.91 versus 0.55 to 0.87). Therefore, a cocktail ELISA containing abundantly expressed antigens enhances the sensitivity of a single antigen and can be a useful diagnostic tool for the detection of active TB.     

Growth of recombinant Mycobacterium tuberculosis H37Ra in mouse macrophages

Mycobacterium tuberculosis H37Rv and H37Ra were derived from the same parental strain but differ strikingly in their virulence for experimental animals. Transfer of genetic material between these closely related strains resulted in the isolation of a number of recombinant H37Ra clones bearing the in vivo growth-promoting ivg locus of H37Rv. The recombinant strain was phagocytosed by murine peritoneal macrophages infected in vivo or in vitro and their intracellular growth rates were compared with the vector control. The intracellular growth of the recombinant was significantly faster than the vector control, but substantially slower than the wild-type H37Rv control, regardless of the method used to infect the macrophages. The slower intracellular growth observed for the recombinant strains was not due to a genetically induced metabolic defect, since they grew in synthetic liquid medium at rates equal to those observed for both H37Rv and H37Ra. Peritoneal macrophage monolayers provide a rapid and convenient assay by which to screen H37Ra recombinants for the presence of putative virulence genes.     

Growth of Virulent and Avirulent Mycobacterium tuberculosis Strains in Human Macrophages

Mycobacterium tuberculosis H37Rv causes progressive disease in animals, whereas the H37Ra strain does not. The relevance of this difference in virulence to human infection is uncertain because these strains have been shown to have similar growth rates in human macrophages. To evaluate the intracellular growth of M. tuberculosis strains in macrophages under conditions similar to those encountered in vivo, we infected human monocyte-derived macrophages with H37Ra, H37Rv, or one of four isolates from tuberculosis patients at a low bacillus-to-macrophage ratio. H37Rv and the patient isolates grew significantly faster than H37Ra, based on the numbers of CFU and acid-fast bacilli. These findings did not result from extracellular mycobacterial growth, differential macrophage viability, or bacillary clumping. In contrast to other published results, these findings indicate that the virulence characteristics of M. tuberculosis strains in animal models are relevant to human tuberculosis infection.     

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

Purpose

Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed.

Materials and Methods

The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains.

Results

The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%).

Conclusion

The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.     

Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain ofMycobacterium tuberculosisin human neutrophils

The phagocytic function of neutrophils is a crucial element in the host defence against invading microorganisms. We investigated phagocytosis and intracellular killing of an attenuated strain ofMycobacterium tuberculosis(H37Ra) by human neutrophils focusing on the role of the cytosolic free calcium concentration [Ca²⁺]_iand certain cytosolic calcium-dependent membrane-binding proteins annexins. Phagocytic uptake did not trigger a calcium rise and occurred independently of different calcium conditions, and in a serum-dependent manner. Changes in the viability of H37Ra were determined by agar plate colony count and a radiometric assay. Neutrophils showed a capacity to kill ingested mycobacteria and this occurred without a rise in [Ca^2+]_i. The ability to kill H37Ra decreased in the absence of extracellular calcium and when intra-extracellular calcium was reduced. Immunofluorescence staining revealed that during phagocytosis of H37Ra, annexins III, IV and VI translocated from cytoplasm to the proximity of the H37Ra-containing phagosomes, whereas the localization of annexin I and V remained unchanged. The translocation of annexin IV occurred even when Ca²⁺-depleted neutrophils ingested H37Ra in the absence of extracellular calcium. We concluded that neutrophil-mediated killing of mycobacteria is a Ca²⁺-dependent process. The fact that the association of certain annexins to the membrane vesicle containing H37Ra differ from other phagosomes suggests a selective regulatory mechanism during phagocytosis of mycobacteria by neutrophils.     

Separation of mycobacterial soluble antigens by isoelectric focusing

Ion-exchange chromatography and gel filtration have been reported to yield partial separation of mycobacterial antigens. These procedures were used in combination with isoelectric focusing in an attempt to purify antigens of Mycobacterium tuberculosis strain H37Ra. The fractionating action of isoelectric focusing is dependent upon differences in the isoelectric points of the proteins to be separated. Culture filtrate of M. tuberculosis H37Ra was chromatographed on Sephadex G-200. This resulted in two widely separated peaks. The first peak, presumably containing high-molecular-weight substances, was then fractionated on a diethylaminoethyl Sephadex anion-exchange column. Three peaks were collected, and each was subjected to isoelectric focusing. Each peak was further separated into two or more fractions. The serological reactivity of each fraction was determined by immunodiffusion and immunoelectrophoresis. Sensitized guinea pigs were also skin-tested with the fractions. Two of the fractions contained only a single precipitinogen. One fraction contained two precipitinogens. A fourth fraction contained three precipitinogens and was also the only fraction to display sensitin activity. Four of the fractions were inactive either as precipitinogens or sensitins. The results suggest that the methods described are useful for the separation of mycobacterial antigens.     

Both B‐1a and B‐1b cells exposed to Mycobacterium tuberculosis lipids differentiate into IgM antibody‐secreting cells

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. The cellular immune response to mycobacteria has been characterized extensively, but the antibody response remains underexplored. The present study aimed to examine whether host or bacterial phospholipids induce secretion of IgM, and specifically anti‐phospholipid IgM, antibodies by B cells and to identify the responsible B‐cell subset. Here we show that peritoneal B cells responded to lipid antigens by secreting IgM antibodies. Specifically, stimulation with M. tuberculosis H37Rv total lipids resulted in significant induction of total and anti‐phosphatidylcholine IgM. Similarly, IgM antibody production increased significantly with stimulation by whole Mycobacterium bovis bacillus Calmette–Guérin. The B‐1 subset was the dominant source of IgM antibodies after exposure to cardiolipin. Both CD5⁺ B‐1a and CD5^? B‐1b cell subsets secreted total IgM antibodies after exposure to M. tuberculosis H37Rv total lipids in vitro. Overall, our results suggest that the poly‐reactive B‐1 cell repertoire contributes to non‐specific anti‐phospholipid IgM antibody secretion in response to M. tuberculosis lipids.     

Dot Blot Assay for Detection of Antidiacyltrehalose Antibodies in Tuberculous Patients

A simple dot blot test with diacyltrehalose (DAT) as the antigen was developed to detect anti-DAT antibodies in tuberculous patients. To enhance antigen-antibody reaction detection, rabbit serum raised against human immunoglobulins was used prior to incubation with a protein A-colloidal gold complex. With the dot blot system, it was possible to obtain a sensitivity similar to that of enzyme-linked immunosorbent assay (ELISA) and a specificity of 97.14%, versus a specificity of 94.29% by the ELISA. We conclude that this simple and fast assay could be used in places where ELISA equipment is not easy available and that it might also be applicable with other Mycobacterium tuberculosis immunogenic antigens.     

Early detection of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> complex in BACTEC MGIT cultures using nucleic acid amplification

We evaluated the application of nucleic acid amplification (NAA) in liquid cultures for the early detection of Mycobacterium tuberculosis. The Cobas TaqMan MTB test, IS6110 real-time PCR, and hsp65 PCR-restriction fragment length polymorphism (RFLP) analysis were used to detect BACTEC MGIT 960 (MGIT) cultures on days 3, 5, 7, and 14. The procedure was initially tested with a reference strain, H37Rv (ATCC 27294). Subsequently, 200 clinical specimens, including 150 Acid Fast bacillus (AFB) smear-positive and 50 AFB smear-negative samples, were examined. The Cobas TaqMan MTB test and IS6110-based PCR analysis were able to detect M. tuberculosis after 1 day when the inoculum of H37Rv was >3 x 10^?2 CFU/ml. After a 5-day incubation in the MGIT system, all three NAA assays had a positive detection regardless of the inoculum size. After a 1-day incubation of the clinical specimens in the MGIT system, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the Cobas TaqMan MTB assay were 70.2%, 100%, 100%, and 82.3% respectively. For IS6110-based PCR analysis, these values were 63.1%, 100%, 100%, and 78.9%, and were 88.1%, 100%, 100%, and 92.1% respectively for hsp65 PCR-RFLP analysis. After a 3-day incubation, the specificity and PPV were 100% for all three NAA tests; the Cobas TaqMan MTB assay had the best sensitivity (97.6%) and NPV (98.3%). The sensitivity, specificity, PPV, and NPV for conventional culture analysis were 98.8%, 100%, 100%, and 99.1%. Thus, NAA may be useful for the early detection of M. tuberculosis after 3 days in MGIT.     

Significance of immune response to Mycobacterium tuberculosis culture filtrate protein antigens in cerebrospinal fluid of tuberculous meningitis patients: A search for diagnostic marker

Mycobacterium tuberculosis (H37Ra) culture filtrate proteins (CFP) are explored as a diagnostic marker for tuberculous meningitis (TBM). Cerebrospinal fluid (CSF) samples from patients were categorized as confirmed (n = 47), suspected (n = 20), and non-TBM (n = 25) cases. Immune response by Western blot revealed TBM CSF samples are having heterogeneous response to CFP. CFP ELISA was 92% sensitive and 38.30% specific. ODs of confirmed TBM and non-TBM cases were significantly different (P < 0.0001) and also the suspected TBM and non-TBM cases (P = 0.0001). No significant difference noticed in TBM and suspected TBM (P = 0.90). Thus, CFP can be a better biomarker for the diagnosis of TBM.     

Lack of protection afforded by ribonucleic acid preparations from Mycobacterium tuberculosis against Mycobacterium leprae infections in mice.

Mycobacterial ribonucleic acid preparations from H37Ra, an attenuated strain of Mycobacterium tuberculosis, provide their usual marked protection against M. tuberculosis challenge; however, they provided no protection against Mycobacterium leprae challenge. Suspensions of intact H37Ra were not effective against M. leprae. Suspensions of BCG gave their usual distinct protection against M. leprae challenge.     

Use of Multiepitope Polyproteins in Serodiagnosis of Active Tuberculosis

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of ~98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of ~93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.     

Analytical Gradient Polyacrylamide Gel-Crossed Line Immunoelectrophores. A Technique for Locating Species Specific Mycobacterium Tuberculosis H37Rv Antigens in Polyacrylamide Gel Columns

Analytical gradient polyacrylamide gel electrophoresis of antigens from Mycobacterium tuberculosis H₃₇RV was combined with crossed line Immunoelectrophoresis to locate the position of antigens in polyacrylamide gel columns that appeared to be specific for M. tuberculosis H₃₇RV. Specificity was established by comparing H₃₇RV with M. kansasii CE in the electrophoretic procedure and the relative mobility (Rm) values are being used in an effort to recover and enrich specific antigens from preparative gel columns. Such fractions should be more suitable in efforts to recover purified monospecific components from M. tuberculosis H₃₇RV.     

The CFP10/ESAT6 complex of Mycobacterium tuberculosis may function as a regulator of macrophage cell death at different stages of tuberculosis infection

Tuberculosis is a human disease caused by infection with Mycobacterium tuberculosis. M. tuberculosis (Mtb) is a facultative intracellular pathogen. The alveolar macrophages provide a critical niche for the intracellular pathogen. It has been shown that virulent strains mycobacteria (Mtb-H37Rv, Mycobacterium bovis) induce less apoptosis in host macrophage than avirulent mycobacteria strains (Bacillus Calmette-Guérin, H37Ra). Comparative genomics analysis has revealed that the region of difference (RD1) of M. tuberculosis is absent from all strains of Bacillus Calmette-Guérin (BCG). On the contrary, it presents in all virulent strains of M. tuberculosis and M. bovis. The culture filtrate protein 10 (CFP10) and early secretory antigenic target protein 6 (ESAT6) are encoded by RD1 genes Rv3874 and Rv3875, respectively. Recent studies indicated that the CFP10 and ESAT6 played an important role in M. tuberculosis virulence. It has been shown that incorporation of the RD1 region into BCG to restore the expression of CFP10 and ESAT6 leads to increasing the virulence and immunogenicity of bacterium. On the contrary, deletion of the genes encoding CFP10 and ESAT6 from the virulent M. bovis strain results in attenuation of virulence. Meanwhile, several studies showed that CFP10 and ESAT6 could inhibit and/or promote the production of tumor necrosis factor α (TNF-α) from macrophages. Furthermore, TNF-α can induce apoptosis and necrosis of infected macrophages in tuberculosis. Considering above results, we hypothesize that the CFP10 and ESAT6 may be involved in the virulence of Mycobacterium through modulating macrophage cell death.     

Serodiagnostic potentialities of enzyme-linked immunosorbent assay (ELISA) using mannophosphoinositides of Mycobacterium tuberculosis H37Rv

The serological response to mannophosphoinositides of Mycobacterium tuberculosis H₃₇Rv and to tuberculin-purified protein derivative (PPD) was examined by enzyme-linked immunosorbent assay (ELISA) in patients suffering from tuberculosis and related diseases. In sputum positive cases 94% samples were found to be positive to mannoside antigens and 77% to PPD, while in sputum negative cases, 71% of samples gave a positive reaction to mannosides and 54% to PPD. The high specificity of mannoside ELISA was demonstrated to be 97% in healthy individuals and 100% in patients suffering from other respiratory diseases, whereas PPD ELISA was 84% and 82% in healthy and infected patients respectively. Thus, ELISA is more specific and sensitive for mannosides than for PPD for the diagnosis of tuberculosis. However, antibodies to mannosides and PPD were detected in lepromatous as well as tuberculoid leprosy patients.