Direct detection from clinical sputum samples to differentiate live and dead Mycobacterium Tuberculosis

Background In this study, we aimed to optimize the condition of propidium monoazide (PMA) treatment for direct detection of Mycobacterium tuberculosis (MTB) from clinical specimens. Methods The light exposure time, dark incubation time, bacterial load, and PMA concentration were varied to determine the optimal condition of PMA treatment. Results Overall, the maximum Delta Cq value was observed in the group receiving a light exposure time of 20 minutes, which was significantly higher than the others (P < 0.05). The prolongation of dark incubation time seemed more likely to result in greater Delta Cq value, and the Delta Cq values were 2.0, 4.1, 6.5, 10.1, and 12.7 cycles under dark incubation time of 10, 20, 40, 60, and 120 minutes, respectively. Alternatively, the 4+ samples exhibited favorable detection results at the application of 10(4)-fold dilution by PMA assay with Cq values higher than 35 cycles. Further evaluation revealed that the PMA assay showed an accordance rate of 98.0% (98/100) among clinical sputa. Conclusions we develop an acceptable method to directly identify the live bacteria from sputum samples. Our data demonstrate that the dark incubation plays a crucial role in the efficacy of PMA treatment for MTB.