Evaluation of polymerase chain reaction for rapid diagnosis of clinically suspected tuberculous pleurisy

Pleural effusion is one of the commonest presentations of tuberculosis, the clinical manifestations being typically abrupt resembling bacterial pneumonia. Since delayed hypersensitivity is the underlying immune response, bacterial load is very low. Owing to these facts, tuberculous pleurisy as an extra-pulmonary disease poses a diagnostic dilemma. The conventional bacteriological methods rarely detect Mycobacterium tuberculosis in pleural fluid and are of limited use in diagnosis of tuberculous pleurisy. We evaluated the efficacy of polymerase chain reaction (PCR) in the diagnosis of tuberculous pleurisy by targeting the gene segment coding for MPB64 protein specific forMycobacterium tuberculosis. Based on the clinical criteria, 82 patients with lymphocytic exudative pleural effusion were included in the study. Patients were analyzed in two groups; one group consisting of 48 patients of tubercular pleural effusion confimed by various diagnostic procedures and another group of 34 patients comprising of non-tubercular pleural effusion. There were no false positive results by PCR and the specificity worked out to be 100%. Twenty two patients tested positive for Mantoux with a sensitivity of 45%. ZN-staining for AFB was found in samples from 15 patients (20% sensitivity). ADA was positive for 28 patients with a sensitivity of 53%. PCR was positive for 32/48 patients (67% sensitivity). Thus, PCR was found to be more sensitive than any other conventional method in diagnosis of clinically suspected tubercular pleurisy.     

Effect of some antitubercular drugs on the calmodulin content ofMycobacterium tuberculosis

The effect of the antitubercular drugs isoniazid (10 μg/ml), ethambutol (10 μg/ml), rifampicin (0.5 μg/ml) and streptomycin (1 μg/ml) on the calmodulin like protein (CAMLP) content ofMycobacterium tuberculosis H₃₇R_v andM. tuberculosis H₃₇R_a was investigated. The drugs were added to actively growing cells at their mid log phase of growth (14 days) and after 12 more hours of incubation, CAMLP was estimated. In both the mycobacteria, all the four antitubercular drugs CAMLP.     

Identification of drug-resistantMycobacterium tuberculosis by single strand conformation polymorphism and cleavase fragment length polymorphism

Twenty isolates ofMycobacterium tuberculosis resistant to rifampicin(RIF), isoniazid(INH) and streptomycin(STR) were analysed by Polymerase Chain Reaction (PCR) amplification of rpoB, katG and rrs genes to evaluate comparative diagnostic significance of genetic assays. Mutations were identified by single strand conformation polymorphism (SSCP) and cleavase fragment length polymorphism (CFLP) and were confirmed by DNA sequencing. SSCP of 4 RIF resistant and 14 INH resistant isolates showed an extra peak at the level of 75-bp and 85-bp respectively, while 2 STR resistant isolates showed 2 peaks with 9 bases difference. CFLP showed a different pattern among RIF, INH and STR sensitive and resistant isolates Thus SSCP and CFLP can be used as alternative diagnostic methods for identification of mutations in RIF, INH and STR resistant strains of M.tuberculosis.     

Detection of tubercular antibody and antigen in sera of bone and joint tuberculosis

Trichloroacetic acid (TCA) solubilized and DEAE fractionatedMycobacterium tuberculosis H₃₇Ra excretory-secretory (ES) antigen viz., Mtb EST DE1 and affinity purified goat antibodies to the TCA solubilized ES antigen (Mtb EST) were explored in detecting tubercular antibody and antigen respectively in sera of bone and joint tuberculosis by indirect and sandwich ELISA. Out of total 36 bone & joint tuberculosis cases, tubercular antibody was detected by indirect ELISA in 30 patients (sensitivity 83%), while circulating tubercular antigen was detected by sandwich ELISA in 27 patients (sensitivity 75%). Out of 34 non tubercular disease control cases, 10 patients showed positive reaction for antibody while only 4 patients showed positive reaction for antigen. In another group of 34 healthy subjects who were screened, 4 individuals showed positive reaction for tubercular antibody and 2 cases for antigen. This study shows that antigen detection assay using affinity purified anti Mtb EST antigen antibody is superior with overall specificity of 91% as compared to antibody detection assay with 75% specificity in bone & joint tuberculosis.