Evaluation of an in-house-developed PCR for the diagnosis of tuberculous meningitis in Indian children.

Early and rapid detection of the causative organism is necessary in tuberculosis, particularly tuberculous meningitis, as the disease affects mainly children and if untreated or improperly treated can cause severe central nervous system disorders and can often be fatal. An in-house-developed PCR technique was developed for the detection of Mycobacterium tuberculosis DNA, in which the target for amplification was a 340 bp nucleotide sequence located within the 38 kDa protein gene. The test can detect as small an amount of DNA as 10 fg, which is equivalent to two to three organisms, and is highly specific. Amplified product was detected by ethidium bromide staining after electrophoresis and Southern hybridization. Evaluation of test sensitivity and specificity was carried out using acid-fast bacilli-positive sputum samples from patients with pulmonary tuberculosis and an equal number of non-tuberculosis patient samples as negative controls. In a double-masked study 30 cerebrospinal fluid samples from tuberculous meningitis patients and 30 samples from non-tuberculous meningitis patients were investigated. Out of the 30 samples 22 were positive by ethidium bromide-stained gel electrophoresis and 27 gave positive results by Southern hybridization. All of the 30 control samples showed negative results. The sensitivity of this PCR was 90 % and specificity, 100 %.

Evaluation of an in-house-developed radioassay kit for antibody detection in cases of pulmonary tuberculosis and tuberculous meningitis.

A radioassay for the detection of antitubercular antibody has been developed. The technique involves the addition of (125)I-labeled Mycobacterium tuberculosis antigen as a tracer, diluted clinical sample (serum or cerebrospinal fluid [CSF]), and heat-inactivated Staphylococcus aureus to capture the antibody, incubation for 4 h, and quantitation of the amount of antibody present in the sample. A total of 330 serum samples from patients with pulmonary tuberculosis and 138 control serum samples from individuals who were vaccinated with M. bovis BCG and from patients with pulmonary disorders of nontubercular origin were analyzed. Also, 26 CSF samples from patients with tuberculous meningitis and 24 CSF samples as controls from patients with central nervous system disorders of nontuberculous origin were analyzed. Sensitivities of 80 and 73% were observed for patients with pulmonary tuberculosis and tuberculous meningitis, respectively, and specificities of 90 and 88% were seen for the two groups of patients, respectively. The sensitivity was lower, however, for human immunodeficiency virus-infected patients coinfected with M. tuberculosis. The control population could be differentiated from the patient population. This assay is rapid and user friendly and, with its good sensitivity and specificity, should benefit the population by providing diagnoses early in the course of disease and, hence, permit the early administration of appropriate chemotherapy.

Mycobacterium tuberculosis 38kDa antigen and its encoding gene-experience in diagnostic applications.

Our experience has revealed that the detection of 38 kDa antigen or antibody to the antigen in various fluids is useful in diagnosis of various mainfestations of tuberculosis. The PCR developed for 340bp sequence of its encoding gene also shows a high degree of sensitivity and specificity. Thus the 38 kDa antigen/antibody combination or the PCR are ideal for development of kits for diagnosis. These immunoassays to be successful, isolation of the 38 kDa antigen in large quantities is essential. Using recombinant DNA technology and expression inE. coli this has been achieved. However, such recombinant antigen does not have the same immunological properties as the native antigen and hence not suitable in immunodiagnosis. To fully realise the potential of the MoAb defined antigens such as the 38 kDa antigen, 19 kDa antigen and others it is essential to devise alternative vector-host systems that help in glycosylation, do not accumulate as inclusion bodies and can be isolated with less damage.

Study of anti-idiotype antibodies to the monoclonal antibody HGT3a and its relation to the 38 kDa antigen of Mycobacterium tuberculosis.

The hypervariable regions of the immunoglobulins which function as the antigen binding sites are capable of provoking an antibody response and are referred to as anti-idiotypic antibodies. Antisera were raised in rabbits against the idiotypes of a mouse monoclonal antibody HGT3a which binds only to the 38 kDa antigen of the M. tuberculosis complex group of organisms. Idiotype specificity in these antisera was determined by dot ELISA, Western blot and solid phase inhibition assays. In vivo administration of this rabbit anti-idiotypic antibody to Swiss mice evoked an anti-anti-idiotypic antibody response, further confirming the internal antigen mimicry by the anti-idiotypic antibodies of the 38 kDa antigenic epitope and its potential use as a surrogate antigen. Antibody response to the anti-idiotypic antibodies in the sera of patients with pulmonary tuberculosis showed significant correlation with the antibody response to the 38 kDa antigen studied in the same clinical samples indicating a close similarity of the 38 kDa antigen of M. tuberculosis and the rabbit anti-idiotypic antibody produced against MoAb HGT3a.

Polymerase chain reaction in the diagnosis of tuberculosis. Comparison of two target sequences for amplification.

Amplification of a 340 bp sequence of the 38 kDa protein gene of Mycobacterium tuberculosis by the polymerase chain reaction has been developed. The sensitivity of this PCR was shown to be 10 fg both by agarose gel electrophoresis and Southern blot hybridisation being equivalent to 2-3 organisms and highly specific to M. tuberculosis and excluding even M. tuberculosis H37Ra and Mycobacterium bovis BCG. Sputum samples from patients with pulmonary tuberculosis gave a positivity rate of 45%. PCR was also performed using pt8 and pt9 primers which amplified a 541 bp sequence of IS6110. 41% of the above samples gave positive amplification. Three samples that were positive for 38 kDa sequence were negative for IS6110.

Detection of antibodies to defined M. tuberculosis antigen (38 kDa) in cerebrospinal fluids of patients with tuberculous meningitis.

Antibodies to the 38 kDa antigen of M. tuberculosis which is serospecific to the tuberculosis complex group of organisms was studied in CSF samples of patients with tuberculosis meningitis. Patients were classified into four groups, viz. post-mortem-proved, culture-proved, clinically suspected and tuberculoma. Anti-38 kDa antibody was detected by ELISA and was positive in 60%, 80% 62.5% and 0%, respectively in the four groups. Controls showed a false-positive detection of 5%. Follow-up of patients was done up to 6 weeks and antibody levels dropped in all the patient groups.

Use of the recombinant 38-kDa antigen of Mycobacterium tuberculosis as an immunogen for specific antisera production.

The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.

T lymphocytes in pulmonary tuberculosis.

T cells and their sub-populations were evaluated with respect to reactive, intermediate and unreactive forms of tuberculosis as classified by Lenzini. Significant CD4 lymphopenia and a reduction of CD4/CD8 ratios were found in patients with reactive tuberculosis. It was observed that there was a B lymphocytosis, CD8 lymphocytosis and a reduction of CD4/CD8 ratio in patients with intermediate and unreactive forms of tuberculosis. The T lymphocytes and CD4 subset were unchanged. There was no significant difference in the lymphocytes and sub-populations among the intermediate and unreactive groups.

A study of tubercular antigen and antibody in childhood tuberculosis.

A radioimmunoassay for the detection of tubercular (TB) antigen (Ag) and antitubercular antibody (Ab) was evaluated for the serodiagnosis of childhood tuberculosis. Children with primary complex, progressive primary complex, miliary tuberculosis, and calcified lung lesions without clinical evidence of active tuberculosis were studied. Significantly elevated levels of TB Ag and TB Ab isolated from the circulating immune complexes were obtained in primary, progressive primary, and miliary tuberculosis patients as compared to controls (P less than 0.01). The majority of patients with calcified lung lesions and without active tuberculosis demonstrated high levels of antibody. It was observed that elevated levels of TB Ag and/or antibodies were present in 54 per cent of patients with primary complex, 94 per cent of patients with progressive disease and 69 per cent of patients with miliary tuberculosis. It is possible that is suspected patients with the above mentioned diseases, a diagnosis can be established by using these techniques.

Radioimmunoscintigraphic approach for the in vivo detection of tuberculomas--a preliminary study in a rabbit model.

Radioimmunoscintigraphic approach could provide a viable non-invasive alternative for the diagnosis of deeply situated tuberculomas. We have evaluated this in a rabbit model constructed to give a characteristic localized tubercular lesion, with complimentary morphological, histological and antigenic profiles. 131I-anti Myobacterium bovis (BCG) antibody was shown to localize at the lesion, where as 131I-bovine serum albumin and 99mTc-red blood cell scans were negative. The clearance of intradermally-injected tuberculous antigen could be traced into ascending lymph nodes using 131I-anti M. bovis (BCG) antibody.

Characterization of serologic and cell-mediated reactivity of a 38-kDa antigen isolated from Mycobacterium tuberculosis.

An antigen of Mycobacterium tuberculosis with an m.w. of 38,000 has been isolated by affinity chromatography using a monoclonal antibody. This antibody bound only to an antigen found in M. tuberculosis and Mycobacterium bovis BCG. The specificity of the antigen was tested in a vertical study by immunodetection on western blots reacted with hyperimmune sera against M. tuberculosis, M. bovis, and 10 other Mycobacterium species. The antigen was detected only by antisera to M. tuberculosis and M. bovis. Specificity in cell-mediated immunity was tested by skin tests in guinea pigs sensitized with M. tuberculosis, Mycobacterium intracellulare, and Mycobacterium kansasii and by lymphocyte proliferation tests. The 38-kDa antigen induced positive skin test reactions regardless of the Mycobacterium species used to sensitize the animal. The ability of the 38-kDa antigen to sensitize for cell-mediated immunity was tested by injecting mice with the 38-kDa antigen and challenging their lymphocytes in vitro with various mycobacterial antigens. Lymphocyte proliferation was observed in the presence of 38-kDa antigen, M. tuberculosis sonicate antigen, and tuberculin purified protein derivative and to M. kansasii and M. intracellulare. The 38-kDa antigen may contain a specific epitope detected by serology, but also contains epitopes that are cross-reactive for cellular immunity.

Radioimmunoassay for detecting Mycobacterium tuberculosis antigen in cerebrospinal fluids of patients with tuberculous meningitis.

A biotin-avidin radioimmunoassay for detecting Mycobacterium tuberculosis antigen has been developed. The assay involves sandwiching mycobacterial antigens from sonicate preparations and cerebrospinal fluids between two antibodies produced in burros and rabbits. The reaction is amplified by using biotinylated antibody to rabbit IgG and 125I-labeled avidin. The assay has a sensitivity of 20 ng/ml and shows less than 5% cross-reactivity with six other mycobacteria. We studied patients with untreated tuberculous meningitis, patients with treated tuberculous meningitis, patients with nonbacterial meningitis, and patients with bacterial meningitis. Antigen was detectable in two of 56 control samples (40 ng/ml). Hence, samples with greater than or equal to 80 ng of antigen/ml were considered positive. In patients with untreated tuberculous meningitis, antigen levels ranged from 20 to 10,000 ng/ml, and 15 (79%) of 19 samples were positive. In the treated group, only two (10%) of 17 samples were positive. This test promises to be a rapid adjunct in the early diagnosis of tuberculous meningitis.

Production, characterization, and species specificity of five monoclonal antibodies to Mycobacterium tuberculosis.

The production and characterization of five monoclonal antibodies to Mycobacterium tuberculosis are described. Specificity of the monoclonal antibodies was tested against other mycobacterial species by an enzyme-linked immunosorbent assay and immunoblots. HGT 3a, an immunoglobulin M (IgM) antibody, recognizes a molecule of 38,000 molecular weight present only in the tuberculosis complex of M. tuberculosis and Mycobacterium bovis BCG. HGT 6, an IgG1 antibody, recognized two molecules with molecular weights of 43,000 and 45,000 and showed limited cross-reactivity. Three other antibodies, HGT 1, HGT 2, and HGT 4, all belonging to the IgG1 type, recognized multiple bands and showed broad reactivity among all mycobacterial antigens tested, Escherichia coli and Nocardia asteroides.

Sensitivity and specificity of enzyme-linked immunosorbent assay in the detection of antigen in tuberculous meningitis cerebrospinal fluids.

A sandwich enzyme-linked immunosorbent assay was developed for its potential utility in the detection of antigen in the cerebrospinal fluid of patients with tuberculous meningitis. Cerebrospinal fluids examined included those from untreated (group Ia) and treated (group Ib) Mycobacterium tuberculosis meningitis, nonseptic central nervous conditions (group II) such as epilepsy, viral meningitis, and tetany, and nonmycobacterial septic meningitis (group III). The average levels of antigens determined and percent positive specimens, respectively, for each group were (group): Ia, 1.8 micrograms/ml and 75% positive; Ib, 0.37 microgram/ml and 36% positive; II, 0.036 microgram/ml and 100% negative; and III, 0.075 microgram/ml and 100% negative. The system developed employed hyperimmune polyclonal antibody raised against M. tuberculosis and Mycobacterium bovis BCG in burros and rabbits. Cross-reactivity by other mycobacterial species was very low; e.g., 5% for M. kansasii and less than 2% for M. intracellulare, M. avium, M. vaccae, and M. fortuitum. The test shows promise as a specific adjunct for the early diagnosis of tuberculous meningitis.