Evaluation of the detection of Mycobacterium tuberculosis with metabolic activity in culture-negative human clinical samples.

Mycobacterium tuberculosis is assumed to remain in a quiescent state during latent infection, being unable to grow in culture. The aim of this study was to evaluate the detection of viable but non-cultivable bacilli with metabolic activity in human clinical samples using a procedure that is independent of the immunological status of the patient. The study was performed on 66 human clinical samples, from patients subjected to routine diagnosis to rule out a mycobacterial infection. Specimens from pulmonary and extra-pulmonary origins were verified to contain human DNA before testing for M. tuberculosis DNA, rRNA and transient RNA by real-time quantitative PCR. Clinical records of 55 patients were also reviewed. We were able to detect viable but non-cultivable bacilli with a metabolic activity in both pulmonary and extra-pulmonary samples. Mycobacterium tuberculosis RNA was detected in the majority of culture-positive samples whereas it was detected in one-third of culture-negative samples, 20% of them showed metabolic activity. Amplifications of the ftsZ gene and particularly of the main promoter of the ribosomal operon rrnA, namely PCL1, seem to be good targets to detect active bacilli putatively involved in latent infection. Moreover, this last target would provide information on the basal metabolic activity of the bacilli detected.