Molecular diagnosis of drug-resistant tuberculosis

The worldwide emergence and spread of multidrug- resistant [MDR] strains of Mycobacterium tuberculosis has adverse effects on tuberculosis [TB] control programs. The goal of this paper is to describe the advances made in the understanding of the molecular basis of M. tuberculosis resistance to first-line anti-TB drugs, and to discuss the potential of molecular methods in early diagnosis of drug-resistant TB. Molecular methods such as DNA sequencing, polymerase chain reaction, DNA hybridization and restriction fragment length polymorphism have been used to identify/detect mutations in gene-encoding proteins or rRNA which are targets for the first-line anti-TB drugs. High level resistance to rifampin [RIF], isoniazid [INH], pyrazinamide [PZA], ethambutol [EMB], and streptomycin [STR] is caused by mutations in rpoB, katG, pncA, embB and rpsL/rrs genes, respectively. The most common mutations conferring high level resistance to RIF, INH, EMB and STR have been identified in rpoB, katG, embB and rpsLgenes, respectively. Molecular methods to detect the most frequent mutations in the gene encoding functions that are targets for first-line anti-TB drugs have provided encouraging results for early diagnosis of MDR-TB

Identification of mycobacterial recombinant antigens recognized by human T cells

The identification of mycobacterial antigens recognized by human T cells is important for the development of new vaccines and diagnostic reagents, and in understanding the mechanisms involved in the pathogenesis of mycobacterial diseases. In this study, Escherichia coli cell lysates, containing mycobacterial recombinant antigens previously identified using antibodies, were tested with peripheral blood mononuclear cells [PBMC], T cell lines and T cell clones to identify the antigens recognized by human T cells. Although the PBMC responded to natural mycobacterial antigens by proliferation, they did not show any specific response to recombinant antigens containing lysates above the background level proliferation induced by control lysates of E coli. With T cell lines raised against whole mycobacteria, the problem of background proliferation was reduced but the specific responses to the lysated containing recombinant antigens were weak. The proliferative response to specific antigens improved by enriching the T cell lines with E. coli lysates containing homologous recombinant antigens. The best response to recombinant antigens was obtained with mycobacterial antigen-specific T cell clones. However, T cell clones did not respond to some of the antigens which induced proliferation of the enriched T cell lines from the same individuals. This could be because of the inherent bias in cloning procedures which may lead to the expansion of selected T cell populations. Our results suggest that enriched T cell lines are best to determine the T cell reactivity of recombinant antigens, but T cell clones are required to demonstrate the existence of species-specific epitopes

Cross reactive mycobacteria and recombinant antigens restore the invitro response of lepromatous T cells to M. leprae antigen

A large number of studies conducted in the past have shown that T cells from lepromatous leprosy patients are specifically anergic to M. leprae antigens in cell mediated immunity assays. In this study, we demonstrate the existence of M. leprae reactive T cells in lepromatous patients by either enriching them with the antigens of crossreactive mycobacteria or testing them with isolated recombinant antigens of M. leprae. Our results confirm the notion that regulatory mechanisms may contribute to the nonresponsiveness of lepromatous T cells to M. leprae antigens