Characterization of rpoB mutations in rifampin-resistant isolates of mycobacterium tuberculosis cultured from the Iranian patients

In this project we investigated the frequency of mutations within the rifampin resistance-determining region [RRDR] of rpoB gene to determine whether this region is useful for molecular detection of rifampin resistant isolates of Mycobacterium tuberculosis from Iranian patients. A set of 25 rifampin resistant and 5 randomly chosen fully susceptible M. tuberculosis complex strains obtained from sputum samples of individual patients were investigated. The M. tuberculosis H37RvT and CDC 1551 standard strains were used as controls. Using the specific primers, the entire RRDR of rpoB gene of selected samples was amplified and sequenced directly. Genetic alterations in the RRDR were present among 96.0% of isolates. The majority of rifampin resistant isolates [72.0%] showed missense mutations in the core region of rpoB that led to substitutions of amino acids at Ser-531 [60.0%], His-526 [16.0%] or Asp-516 [8.0%]. While the codon 531 has been the most common site of nucleotide substitutions worldwide, the frequencies of mutations at the codons 526 and 516 among the Iranian isolates were different from other geographical regions. Mutation at codon 533 was found at higher frequency [8%] comparing to the report from other countries. The high rate of mutations within the RRDR of the rpoB gene suggests that targeted screening of the RRDR may be feasible for the determination of rifampin resistance in clinical isolates of Mycobacterium tuberculosis from Iran

Purification and molecular analysis of BCG antigen 60

Tuberculosis remains as an important socioeconomical and medical problem throughout the world and especially in Iran. Early and timely diagnosis of pulmonary and extrapulmonary tuberculosis is vital to initiate prompt treatment. Current diagnostic methods are either slow or lack enough sensitivity or specificity. Several mycobacterial antigens are involved in the complex interaction with the immune system of the host. Their identification is important for both diagnosis and protection against mycobacteria. Antigen 60 [A60] is a thermostable antigen found in the cytosol of M. bovis and M. tuberculosis. An ELISA test using A60 is designed for diagnosis of tuberculosis with satisfactory results. In previous studies, A60 has also showed a protective effect against experimental infections and useful immunotherapeutic effects in promotion of cancer development. In the present work we tried to purify A60 from the cytoplasm of BCG. A60 was purified by exclusion gel chromatography using sepharose 4B. A60 was recognized by bidimensional immunoelectrophoresis with anti-BCG and anti-A60 antiserum, where it appears as the less mobile component. In agarose electrophoresis, A60 showed only one band but in immunodiffusion it showed two immunoprecipitinogen lines with anti-BCG anti-serum. In analyzing with dot blotting, both cytoplasm and cell wall of BCG showed positive reaction with anti-A60 anti-serum. When A60 was fractionated by SDS-PAGE and analyzed by western blot using anti-A60 antibody, 65,46,40, 38 and 35 KDa protein fractions were identified. It is concluded that A60 is a macromolecular antigen of BCG with a molecular weight of 10[6]-10[7] Da and is a lipoprotein-polysaccharide complex which contains several proteins. A60 is present in both cytoplasm and cell wall of BCG and can easily be purified from BCG vaccine using exclusion chromatography by sepharose 4B, to be used for designing diagnostic tests for TB