Quantitative analysis of mRNA as a marker for viability of Mycobacterium tuberculosis

Numerous assays which use conserved DNA or rRNA sequences as targets for am plification have been described for the diagnosis of tuberculosis. However, these techniques have not been applied successfully to the monitoring of t herapeutic efficacy owing to the persistence of amplifiable nucleic acid be yond the point at which smears and cultures become negative. Semiquantitati ve analysis of rRNA has been used to reduce the time required for antimicro bial susceptibility testing of Mycobacterium tuberculosis, although growth for up to 5 days in the presence of some drugs is still required to discrim inate resistant strains. The purpose of the present study was to determine whether quantitative analysis of M. tuberculosis mRNA could be used to asse ss bacterial viability and to illustrate the application of this technique to rapid determination of drug susceptibility. Levels of mRNA encoding the 85B protein (alpha-antigen), IS6110 DNA, and 16S rRNA were compared in para llel cultures of M. tuberculosis that were treated with either no drug, 0.2 mu g of isoniazid per ml, or 1 mu g of rifampin per mi. Exposure of sensit ive strains to isoniazid or rifampin for 24 h reduced the levels of 85B mRN A to <4 and <0.01%, respectively, of those present in control cultures with out drug. In contrast, the levels of IS6110 DNA and 16S rRNA did not dimini sh over the same period. Strains which were resistant to either isoniazid o r rifampin demonstrated no reduction in 85B mRNA in the presence of the dru g to which they were nonresponsive. Quantitative analysis of 85B mRNA offer s a potentially useful tool For the rapid determination of M. tuberculosis drug susceptibility and for the monitoring of therapeutic efficacy.