Quantitative Analysis of mRNA as a Marker for Viability of Mycobacterium tuberculosis

Abstract

ABSTRACT Numerous assays which use conserved DNA or rRNA sequences as targets for amplification have been described for the diagnosis of tuberculosis. However, these techniques have not been applied successfully to the monitoring of therapeutic efficacy owing to the persistence of amplifiable nucleic acid beyond the point at which smears and cultures become negative. Semiquantitative analysis of rRNA has been used to reduce the time required for antimicrobial susceptibility testing of Mycobacterium tuberculosis , although growth for up to 5 days in the presence of some drugs is still required to discriminate resistant strains. The purpose of the present study was to determine whether quantitative analysis of M. tuberculosis mRNA could be used to assess bacterial viability and to illustrate the application of this technique to rapid determination of drug susceptibility. Levels of mRNA encoding the 85B protein (α-antigen), IS 6110 DNA, and 16S rRNA were compared in parallel cultures of M. tuberculosis that were treated with either no drug, 0.2 μg of isoniazid per ml, or 1 μg of rifampin per ml. Exposure of sensitive strains to isoniazid or rifampin for 24 h reduced the levels of 85B mRNA to <4 and <0.01%, respectively, of those present in control cultures without drug. In contrast, the levels of IS 6110 DNA and 16S rRNA did not diminish over the same period. Strains which were resistant to either isoniazid or rifampin demonstrated no reduction in 85B mRNA in the presence of the drug to which they were nonresponsive. Quantitative analysis of 85B mRNA offers a potentially useful tool for the rapid determination of M. tuberculosis drug susceptibility and for the monitoring of therapeutic efficacy.