ABSTRACT
OBJECTIVE: To evaluate the significance of quantitative analysis of Mycobacterium tuberculosis mRNA in the test of susceptibility of Mycobacterium tuberculosis strains to rifampin. METHODS: The susceptibility to rifampin of fifty-three clinical isolated strains was test by the percentage of Mycobacterium tuberculosis 85B mRNA copies before and after the use of rifampin, and 1% and 10% were used as the standards for drug resistance, which was compared with the absolute concentration method. Among them, 29 were rifampin resistant strains and 24 rifampin sensitive strains. RESULTS: When the concentration of rifampin was 1 micro g/ml, and 1% and 10% were used as the standards, the accuracy of quantitative analysis of Mycobacterium tuberculosis mRNA with the absolute concentration method was 70% and 81% respectively, and the sensitivity of quantitative analysis of Mycobacterium tuberculosis mRNA was 100% and 100% respectively, while the specificity of quantitative analysis of Mycobacterium tuberculosis mRNA was 33% and 58%. When the concentration of rifampin was 2 micro g/ml, the accuracy of quantitative analysis of mRNA with the absolute concentration method was 85% and 93% respectively, and the sensitivity was 100% and 97% respectively, while the specificity was 67% and 88% respectively. When the concentration of rifampin was 4 micro g/ml, the accuracy was 93% and 93%, and the sensitivity was 93% and 93%, while the specificity was 92% and 92%. CONCLUSIONS: Quantitative analysis of Mycobacterium tuberculosis mRNA was a rapid, sensitive method in rifampin resistance screening. We suggest that the critical concentration of rifampin be 2 micro g/ml, and the critical proportion of mRNA copy be 10%.
Subject(s)
Antibiotics, Antitubercular/pharmacology , Drug Resistance/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Rifampin/pharmacology , Biomarkers , Microbial Sensitivity Tests/methods , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Tuberculosis, Multidrug-Resistant/microbiologyABSTRACT
OBJECTIVE: To study whether quantitative analysis of M. tuberculosis mRNA could be used to assess bacterial viability. METHODS: The levels of M. tuberculosis mRNA were compared 24, 48, and 72 hours after M. tuberculosis H(37)R(V) was treated with no drug, 1.0 micro g/ml and 10.0 micro g/ml rifampicin, 0.2 micro g/ml and 1.0 micro g/ml isoniazid, 2 micro g/ml and 4 micro g/ml ethambutol, 2 micro g/ml and 4 micro g/ml streptomycin, 1.25 micro g/ml and 5.00 micro g/ml ofloxacin. The levels of mRNA were determined by quantitative PCR. RESULTS: After exposure to isoniazid, rifampicin, and streptomycin for 24 h, the level of M. tuberculosis H(37)R(V) was reduced markedly. Similarly, after exposure to isoniazid, streptomycin, ethambutol, and ofloxacin for 72 h, the level of M. tuberculosis H(37)R(V) was identical, but higher than that after exposure to rifampicin. Exposure of M. tuberculosis H(37)R(V) to rifampicin for 24 h reduced the levels of 85B mRNA to 0.02% of the control; exposure to other drugs for 24 h reduced the levels to 1% approximately 10% of the control, and for 72 h to less than 1% of the control. CONCLUSIONS: The levels of mRNA can show the difference between M. tuberculosis H(37)R(V) exposed and unexposed to drugs, and are consistent with colony forming unit. mRNA could be used as a marker for assessing the viability of M. tuberculosis.
