Long-term persistence of BCG Pasteur in lungs of C57BL/6 mice following intranasal infection.

Different Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine substrains may vary in their efficacy. Here, we describe differences in disease progression and pathology in the lungs of female C57BL/6 mice infected intranasally with BCG Russia or BCG Pasteur and followed for 17 months. The lungs were investigated for bacillary load, histopathology and expression of cytosolic and secreted proteins by immunohistochemistry. BCG Russia was cleared from the lungs by 8 months. BCG Pasteur reached a low-level persistence at 8 months and remained at this level until the end of the experiment. BCG Pasteur induced greater pathology than BCG Russia, and there were more macrophage and lymphocyte infiltrates in animals infected with BCG Pasteur (P < 0.05). Bacterial growth correlated with cellular infiltration. All selected mycobacterial proteins were found to be expressed in the lesions by both BCG strains, and there were only minor variations between the strains. Furthermore, we identified isolated cells containing a high mycobacterial protein load in the normal-looking lung parenchyma. The infected cells in the healthy areas of the lung may represent the ability of mycobacteria to evade immune activation and thereby persist in the host. Clearance of BCG Russia indicates that a more effective and sterilizing immune response is established by this strain. On the other hand, the ability of BCG Pasteur to persist could be important for long-term protection against challenge with Mycobacterium tuberculosis.

MPB70 and MPB83--major antigens of Mycobacterium bovis.

MPB70 and MPB83 are among the most studied mycobacterial antigens. They are highly homologous proteins within Mycobacterium tuberculosis complex members, and the orthologs in different members of the complex are virtually identical. They are major antigens highly expressed by Mycobacterium bovis and considerably less abundantly expressed by M. tuberculosis. There are two genes encoding these proteins within an operon of six genes. MPB70 and MPB83 are encoded as precursor proteins with typical signal peptides for export through the general secretory pathway. MPB70 is a soluble secreted protein cleaved by signal peptidase I, while MPB83 is a glycosylated lipoprotein processed by signal peptidase II and located at the surface, possibly with the lipid tail coupled to the N-terminal cysteine embedded in the mycobacterial outer membrane. The expression of these genes is controlled by the transcriptional regulator SigK, and a point mutation in SigK explains why some Bacille Calmette-Guérin (BCG) strains express only minute amounts of MPB70 and MPB83. BCG strains that have retained the high expression of MPB70 and MPB83 of wild type M. bovis are linked to a risk of BCG osteitis in <1-year-old children as a complication to BCG vaccination at birth. The proposed mechanism for this is based on homology of these proteins with osteoblast-specific factor II which is a homofilic adhesion protein expressed on osteoblasts in growing bone tissue. T-cell responses and antibody responses to these proteins have been extensively explored for sensitive and specific diagnosis of bovine tuberculosis.

HIV induces both a down-regulation of IRAK-4 that impairs TLR signalling and an up-regulation of the antibiotic peptide dermcidin in monocytic cells.

HIV-infected individuals have an increased risk of invasive bacterial infections, even at early clinical stages with relatively normal CD4(+) T-cell counts. The pathogenic mechanisms behind this are not fully understood. However, an increasing number of studies indicate that HIV may impair the innate immunity to bacteria by infecting key cells of the monocyte/macrophage lineage. In this study, the effects of HIV infection on the protein profile of undifferentiated monocyte-like THP-1 cells were examined by a mass spectrometric approach based on stable isotope labelling with amino acid in cell culture (SILAC). We identified 651 proteins, of which nine proteins were down-regulated and 17 proteins were up-regulated in HIV-infected THP-1 cells as compared to uninfected controls. Most remarkably, the IL-1 receptor associated kinase 4 (IRAK-4), which is essential for virtually all TLR signalling, was suppressed, whereas the precursor for the antibiotic peptide Dermcidin was up-regulated in HIV-infected cells. Upon stimulation of either TLR2 or TLR4, the HIV-infected THP-1 cells displayed reduced TNF-alpha secretion. The HIV-induced down-regulation of IRAK-4 was reconfirmed in monocyte-derived macrophage cell cultures. These data suggests that HIV may impair the TLR signalling cascade for pathogen recognition in cells of the monocyte/macrophage lineage and thus, may reduce the ability of the innate immune system to sense invading pathogens and initiate appropriate responses.

Antigen analysis of Mycobacterium tuberculosis H37Rv culture filtrate proteins.

Culture filtrates of Mycobacterium tuberculosis H37Rv highly enriched with secreted proteins were used to identify antigens recognized by a serum pool from tuberculosis patients. Two different approaches were used to separate the culture filtrate protein mixture: (i) proteins were fractionated according to their hydrophobicity using an HPLC-C18 chromatography column followed by separation based on their molecular mass by SDS-PAGE and subsequent immunoblotting or (ii) proteins were separated by two-dimensional gel electrophoresis, based on their isoelectric point and their molecular mass. Twenty serologically reactive proteins were ultimately identified by both methods, including four novel antigens. Further, to estimate the immunogenicity of the identified culture filtrate proteins, the relative antibody quantities were measured using Image master software. Our results show that the antibodies against proteins belonging to the antigen 85 complex were the most abundant in the serum of patients with active tuberculosis. The most immunogenic proteins in terms of high antibody-to-protein-ratio were Rv3881c and three lipoproteins Rv0934 (the 38 kDa antigen), Rv0932c (pstS2), and Rv3006 (LppZ). Rv 3881c [corrected] is located close to the RD1 region and is also present in BCG. [corrected] The proteins from the M. tuberculosis H37Rv culture filtrate are strong candidates to be evaluated for improvement of the serodiagnostic tests of tuberculosis.

Antibody response to long-term and high-dose mould-exposed sawmill workers.

Exposure to moulds is thought to cause adverse health effects ranging from vague subjective symptoms to allergy and respiratory diseases. Until now, most studies have been emphasizing low levels of exposure. In Norwegian sawmills during the 1980s, extensively high spore counts up to 10(7) spores/m3 air were reported. By using serum samples obtained from sawmill workers during that period, in addition to control sera, we studied the antibody response of all classes and IgG subclasses to Rhizopus microsporus at different levels of exposure. Antigen specificity was further studied by Western blotting. Exposure to R. microsporus was accompanied by R. microsporus-specific antibody production against a wide range of antigenic components most likely of both protein and carbohydrate nature. Increasing levels of mould-specific IgG1, IgG2, IgG4 and IgA antibodies were associated with increased exposure, while the highest levels of exposure were associated with a somewhat reduced level of mould-specific IgE antibodies. In conclusion, the present study strongly suggests that high mould exposure can induce a strong IgG and IgA response in a dose-dependent manner.

Correlating sequential homology of Mce1A, Mce2A, Mce3A and Mce4A with their possible functions in mammalian cell entry of Mycobacterium tuberculosis performing homology modeling.

OBJECTIVE: The striking homology of the Mycobacterium tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) with other mycobacterial species and the proposed role of the mammalian cell entry protein 1A (Mce1A) of M. tuberculosis to facilitate invasion of host cells have led us to look into the finer details of these proteins in order to better understand their structure-function relationship. DESIGN: We performed sequential alignments and secondary structure predictions of Mce1A, Mce2A, Mce3A and Mce4A, and compared these results with results from homology modeling by fold prediction and threading. RESULTS: Sequential alignments showed that Mce1A and Mce2A are highly homologous, close to 70%, while the other combinations gave only about 30% similarities. The major parts of the proteins aligned without gaps and there were striking similarities by secondary structure predictions indicating that the proteins would have similar folds and to be alpha/beta proteins like the previously reported Mce1A model based on Colicin N. Fold prediction showed that the best templates for Mce2A were substrate-binding domain of DnaK and slow processing precursor penicillin acylase from Escherichia coli while the alpha-domains of Mce3A and Mce4A could both be modeled using the cytoplasmic domain of serine chemotaxis receptor as template. CONCLUSION: Although different templates had to be used to model the MceA proteins, functional information may be derived that is relevant for their overall function in M. tuberculosis. The beta-domain is probably involved in binding with the receptors on target cells while the alpha-domain is more likely to be involved in pore formation. As predicted from the folds, Mce3A and Mce4A model structures indicate a lipid bound conformation and therefore may be required in signaling events of the mammalian cell entry process.

Comparison of antigens and antisera in crossed immunoelectrophoresis by dual dilution.

The localization of individual precipitates in the crossed immunoelectrophoresis (CIE) pattern is an important feature for their identification, but in a given system this pattern may vary markedly when different antigen preparations or antisera are used. Two different antigen preparations or antisera may be compared by mixing them in various proportions in a set of four CIE plates. The amounts of the preparations to be compared are selected to obtain a gradual change in position of individual precipitates if differences are encountered. Similarities between antigen or antibody preparations are recorded when the positions of precipitates remain unchanged. The technique is particularly valuable for comparing complex patterns with a large number of precipitate lines.

Diffusion blotting for rapid production of multiple identical imprints from sodium dodecyl sulfate polyacrylamide gel electrophoresis on a solid support.

A simple and fast method for diffusion blotting of proteins from precast SDS-PAGE gels (0.5 mm) was developed. The efficiency of protein transfer was evaluated using 14C labelled proteins. Diffusion blotting for three minutes, gave a transfer of 10% compared to electroblotting. By doubling the transfer time for each subsequent imprint, four imprints were made from the same lane with similar amounts of protein transferred onto each imprint. With a transfer time of three minutes each, it was possible to obtain at least ten imprints with all the proteins visible in all the imprints. There was no detectable loss in resolution as compared to electroblotting. The method also works well with an immuno-detecting system. The number of imprints which can be obtained, is dependent on the sensitivity of the detection system and the amount of protein applied. The greatest advantage of diffusion blotting compared to electroblotting is that several imprints can be made from each lane, and different antisera can be tested on identical imprints. The gel remains on its plastic support which prevents it from stretching and compression; this ensures identical imprints and facilitates more reliable molecular mass determination. If only a few imprints are made, sufficient protein remains within the gel for general protein staining. These advantages make diffusion blotting the method of choice when quantitative protein transfer is not required.

Integration of monoclonal antibodies in quantitative immunoelectrophoresis by indirect immunoprecipitation.

Characterization of the specificity of monoclonal antibody in crossed immunoelectrophoresis has been achieved by mixing the monoclonal antibody with mycobacterial antigen in the circular antigen well. After electrophoresis in the first dimension, the separated antigens and antigen complexed with monoclonal antibody were run into an intermediate gel containing rabbit anti-mouse immunoglobulin and a top gel with polyvalent rabbit anti-BCG immunoglobulin. Monoclonal antibody with bound antigen precipitated in the intermediate gel while the other antigens precipitated in their reference positions. The method was simple, efficient and sensitive. Selected monoclonal antibodies were used to demonstrate the characteristic features of the method. In rocket immunoelectrophoresis the rocket height of a monoclonal antibody may be significantly altered by adding the relevant antigen. This principle can be exploited when the polyvalent antibodies do not precipitate the antigen, and it may be used for efficient screening of monoclonal hybridoma culture supernatants. This approach may permit the quantitation of an antigen when only monoclonal antibody is available.

Extensive sequence homology between the mycobacterium leprae LSR (12 kDa) antigen and its Mycobacterium tuberculosis counterpart.

The Mycobacterium leprae LSR (12 kDa) protein antigen has been reported to mimic whole cell M. leprae in T cell responses across the leprosy spectrum. In addition, B cell responses to specific sequences within the LSR antigen have been shown to be associated with immunopathological responses in leprosy patients with erythema nodosum leprosum. We have in the present study applied the M. leprae LSR DNA sequence as query to search for the presence of homologous genes within the recently completed Mycobacterium tuberculosis genome database (Sanger Centre, UK). By using the BLASTN search tool, a homologous M. tuberculosis open reading frame (336 bp), encoding a protein antigen of 12.1 kDa, was identified within the cosmid MTCY07H7B.25. The gene is designated Rv3597c within the M. tuberculosis H37Rv genome. Sequence alignment revealed 93% identity between the M. leprae and M. tuberculosis antigens at the amino acid sequence level. The finding that some B and T cell epitopes were localized to regions with amino acid substitutions may account for the putative differential responsiveness to this antigen in tuberculosis and leprosy.

Searching for secreted proteins of Mycobacterium leprae.

In mycobacteria secreted proteins represent a distinct group, probably of particular importance for development of immune responses following infection. Quantification of individual proteins in Mycobacterium tuberculosis culture fluid and corresponding disrupted bacilli permits determination of a localization index for identification of secreted proteins. This procedure cannot be applied for Mycobacterium leprae since secreted proteins are lost during isolation of bacilli from tissues. The DNA sequences of secreted proteins of M. tuberculosis were compared with sequences of M. leprae. Genes for homologues of the 85a, 85b, 85c, mpt32 (apa), mpt51, erp, mtc28, mtb12, Rv3354 and Rv0526 genes were identified. All of these and six genes of the mcel operon contain signal sequences for secretion in M. leprae as well. In several instances the local distance between marker genes and occurrence on the same or the complementary DNA strand was similar in these two species. The genomic organization of genes for secreted proteins is thus very similar in M. leprae and M. tuberculosis, the homology being higher for the mature polypeptide chains than for the corresponding signal peptides.

Progress in serodiagnosis of Mycobacterium tuberculosis infection.

One-third of the world population is estimated to have Mycobacterium tuberculosis infection. Accurate and timely identification of infected individuals is critical for treatment and control. The current diagnostic methods lack the desired sensitivity and specificity, require sophisticated equipment and skilled workforce or take weeks to yield results. Diagnosis of extrapulmonary TB, TB-HIV co-infection, childhood TB and sputum smear-negative pulmonary TB pose serious challenges. Interest in developing serodiagnostic methods is increasing because detection of antibody is rapid, simple and relatively inexpensive, and does not require a living cell for detection. Three types of tests, namely screening tests to overcome diagnostic delay, specific tests for diagnosis of extrapulmonary TB and other bacteriologically negative cases, and tests for vaccine-induced immunity need critical consideration. Several factors must be considered to develop serodiagnostic methods for TB. Antigen recognition by infected individuals is highly heterogeneous due to stage of disease, differences in HLA types, strain of the bacilli, health of the patient and bacillary load. With advances in molecular biological techniques, a number of novel antigens have been identified. Some of these antigens have proven valuable in detecting specific antibodies in some of the most challenging TB patients. The best example is a fusion protein containing several M. tuberculosis proteins (e.g. CFP-10, MTB8, MTB48, MTB81 and the 38-kDa protein) which showed encouraging results in detecting antibodies in sera of patients, including TB-HIV co-infection. This review presents progress made in the serodiagnosis of TB during the last decade.

Reduced apoptosis and increased inflammatory cytokines in granulomas caused by tuberculous compared to non-tuberculous mycobacteria: role of MPT64 antigen in apoptosis and immune response.

Inhibition of apoptosis of infected macrophages by pathogenic mycobacteria is suggested to be an important virulence mechanism, but little is known about the mycobacterial proteins involved in the inhibition of apoptosis. In this study we investigated differences in apoptosis and immune response and their correlation with the expression of Mycobacterium tuberculosis complex-specific secretory protein MPT64 in lesions caused by tuberculous or non-tuberculous mycobacteria by analysing the in situ expression of apoptosis-related proteins (FasL, Fas, Bax, Bcl-2), apoptotic cells, inflammatory cytokines [tumour necrosis factor (TNF)-alpha, interleukin (IL)-10, transforming growth factor (TGF)-beta, interferon (IFN)-gamma] and MPT64 antigen. The discrimination of mycobacteria was made by nested polymerase chain reaction (PCR) amplification of IS6110, which is specific for M. tuberculosis complex organisms. Forty-seven cases of lymphadenitis with necrotic granulomas were evaluated. With nested PCR, 30/47 cases were positive for M. tuberculosis. MPT64 antigen was detected specifically in the PCR-positive cases. Granulomas caused by tuberculous mycobacteria had fewer apoptotic cells, higher numbers of cells expressing TNF-alpha and TGF-beta and less extensive necrosis than granulomas caused by non-tuberculous mycobacteria. There was a significant negative correlation between apoptotic cells and the number of cells expressing MPT64 antigens, suggesting a role for MPT64 protein in the inhibition of apoptosis. Granulomas with higher amounts of MPT64 also showed a greater number of cells expressing TGF-beta than those with lower amounts of MPT64. In conclusion, this study supports the hypothesis that inhibition of apoptosis is a virulence mechanism for tuberculous mycobacteria. Correlation of MPT64 antigen with expression of macrophage deactivating cytokines and reduced apoptosis suggests its role in pathogenesis and bacillary persistence.

Vaccine approaches to prevent tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is one of the main killers among infectious pathogens in the world and represents an important factor that sustain poverty in developing countries. Failure of the BCG vaccine to protect in endemic regions, and increasing problems with multi-drug-resistant TB calls for development of better vaccines to prevent reactivation of tuberculosis. It has been estimated that an effective post-exposure vaccine will prevent 30-40% of the TB cases. New vaccines should also prevent development of TB in HIV-infected individuals. Recent characterization of M. tuberculosis H37Rv by proteomic methods has revealed a large number of novel secreted proteins that should be investigated in mouse models for latent and slowly progressive TB. There is an important balance between control of infection and tissue destruction in TB, and M. tuberculosis has developed strategies to prevent immune-mediated sterilization. Central to this strategy is inhibition of apoptosis of macrophages. Development of novel vaccines should therefore take into consideration the effects on central markers to obtain a better picture of regulation of immunity, including FasL and Bcl-2 which are essential in regulation of apoptosis.

Immunoglobulin G antibodies against environmental moulds in a Norwegian healthy population shows a bimodal distribution for Aspergillus versicolor.

Immunoglobulin G (IgG) antibodies against moulds related to indoor dampness problems are used as biomarkers to indicate exposure. In the present study, we evaluated the frequency of mould exposure in an adult healthy population by examining levels of mould-specific IgG antibodies in Norwegian blood donors. Using enzyme-linked immunosorbent assay, 106 blood donor sera were analyzed for IgG antibodies to Aspergillus versicolor, Penicillium chrysogenum, Cladosporium herbarum, Stachybotrys chartarum and Fusarium oxysporum. The levels of specific IgG antibodies to P. chrysogenum, C. herbarum and S. chartarum correlated (r = 0.46-0.62). Responses to A. versicolor were considerably stronger than to the other moulds, and another 996 blood donor sera were analyzed for IgG antibodies to this mould. Women had significantly higher levels of specific IgG antibodies to A. versicolor than men. The concentration of A. versicolor-specific IgG antibodies showed a non-Gaussian, bimodal distribution profile, in which 12.5% were defined as positive to exposure. This suggests that significant mould exposure in a healthy population can be calculated from mean + 1SD. Western blotting analyses showed that antibody responses to A. versicolor were largely directed against carbohydrate antigens of unknown saccharides.

Allergic sensitisation in tuberculosis and leprosy patients.

BACKGROUND: A negative association has been observed between infections and allergy in several studies. The aim of the present study was to examine whether tuberculosis and leprosy patients have more or fewer allergies than healthy individuals. METHOD: Sera from tuberculosis patients, leprosy patients and healthy controls were analysed by ELISA and Pharmacia Unicap for serological markers for allergy and mycobacterial infection. The serological markers for allergy were total IgE, specific IgE using Phadiatop and specific IgE to the dust mite allergen Dermatophagoides pteronyssinus 1 (Der p 1). Serological markers for mycobacterial infections included specific IgG to a mixture of bacille Calmette-Guérin culture filtrate antigens, to purified mannose-capped lipoarabinomannan (manLAM) and to purified secreted antigen 85B. RESULTS: Both tuberculosis and leprosy patients had significantly higher levels of total IgE than controls. Furthermore, a significantly higher level of specific IgE (Phadiatop) was also found in the tuberculosis patients compared with controls. A similar result, but not statistically significant, was observed for the leprosy group. Specific IgG to antigen 85B and to manLAM was found to be significantly higher in both tuberculosis and leprosy patients compared with controls. In addition, leprosy patients had significantly more IgG to the BCG culture filtrate antigen than controls. CONCLUSIONS: The results indicate that patients with mycobacterial infections have allergic sensitisation more frequently compared with healthy controls. This is seemingly in contrast with the notion that there is a negative association between allergy and infection ('hygiene hypothesis'). However, since only one in ten of those infected with Mycobacterium tuberculosis will develop the disease, patients with active mycobacterial disease represent a selected group. A similar relationship applies for leprosy. It is conceivable that those predisposed to allergy are less resistant to mycobacterial infections.

Liberation of soluble proteins from live and dead mycobacterial cells and the implications for pathogenicity of tubercle bacilli hypothesis.

Soluble proteins liberated from live M. tuberculosis are translocated through the cytoplasmic membrane to a 'periplasmic space'. For further export of proteins across the outer permeability barrier, it is necessary to postulate an excretion mechanism possibly involving some kind of porin. Observations of the repertoire of proteins in culture filtrates after liquid culture of M. tuberculosis show that a large repertoire of various kinds of proteins cross the outer permeability barrier of tubercle bacilli indicating that the excretion mechanism has a wide range of specificities for proteins. Culture filtrates of tubercle bacilli almost always contain both truly secreted proteins and cytoplasmatically-derived proteins. It is questionable whether cytoplasmic proteins can cross an intact cytoplasmic membrane. The simplest explanation for the appearance of cytoplasmic proteins in culture filtrates of tubercle bacilli would be that they are released after disintegration of the cytoplasmic membrane in dying or dead bacilli. Tubercle bacilli armed with secreted factors that may specifically inhibit innate and adaptive immune responses, excrete these from the periplasmic space of live bacilli. Unspecific in its character, the excretion mechanism also liberates proteins that are essential for building and maintaining the cell wall, thereby reducing the effectiveness of this process. This may be part of the explanation why M. tuberculosis and other pathogenic mycobacteria grow so slowly. Finally, it may be postulated that dormant or latent tubercle bacilli use their repertoire of secreted proteins to control their intracellular habitat and that bacterial cytoplasmic proteins would not be liberated from such bacilli. The consequence would be that only immune responses to secreted proteins would be effective for elimination of the dormant stage of infection. In a situation with active infection there will be considerable growth and turnover of bacilli with liberation of all kinds of immunogenic substances from the bacilli. In this situation immunity against cytoplasmic proteins would also be effective and immunity to cytoplasmic proteins should also be effective for control of the reactivation of latent disease because as soon as the bacilli start to grow there will also be a subpopulation of dead bacilli on the arena.

The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis.

The large number of different proteins synthesized by the mycobacterial cell are currently classified and studied in terms of groups of proteins with certain common properties such as physical and chemical characteristics, function, and localization in the mycobacterial cell. Proteins that are actively secreted during culture on synthetic media represent a particular group of great current interest. At least eight proteins secreted by Mycobacterium tuberculosis have been isolated and characterized to various extents. The genes coding for five proteins secreted from M. tuberculosis and/or Mycobacterium bovis BCG have been cloned and sequenced. All of them contain typical signal sequences. The proteins of the antigen 85 complex, which form the main subject of this review, are often the most common proteins in M. tuberculosis culture fluid. The constituents denoted 85A, 85B, and 85C are encoded by three genes located at different sites in the mycobacterial genome and show extensive cross-reactivity as well as homology at amino acid and gene levels. The proteins differ slightly in molecular mass in the 30- to 31-kDa region, and all of them are fibronectin-binding proteins, but the significance of the latter observation and the role of these proteins in mycobacterial physiology and interaction with the infected host remain to be elucidated. The antigen 85 complex proteins are strongly immunogenic in natural and experimental mycobacterial infections in terms of both induction of antibody synthesis and T-cell-mediated reactions. The well-recognized difference in the efficacy of live and dead mycobacterial vaccines should be considered in relation to the group of secreted antigens. After inoculation, live bacteria in vaccines such as BCG multiply in the host, probably releasing several constituents belonging to the class of secreted proteins and hence resulting in more efficient stimulation of the immune system. Secreted mycobacterial antigens are expected to be of particular significance in induction of various immune responses that are responsible for development of protective immunity in some individuals and for clinical symptoms and complications of the ensuing disease in others.

The 38-kDa protein of Mycobacterium tuberculosis: a review.

This review illustrates that the 38-kDa protein is one of the most important antigens of Mycobacterium tuberculosis. It is actively secreted but partly attached to the surface of the mycobacterial cell by a lipid tail that may also be responsible for binding of carbohydrate to the protein. It is a major constituent of M. tuberculosis culture fluid after growth on the synthetic Sauton medium and occurs in bacille Calmette-Guérin in far lower concentrations. The protein induces B and T cell responses with high specificity for infection with M. tuberculosis and is a prime candidate for development of new diagnostic reagents for tuberculosis.