To the Editors:
Progress made to improve laboratory capacity for tuberculosis (TB) diagnosis led to the development of molecular assays that are now replacing conventional microscopy and culture-based methods on a large scale [1, 2]. Unfortunately, current molecular techniques detect both live and dead bacteria, and a positive result does not imply the viability of the pathogen. Indeed, DNA can persist for a long period after bacterial death and nucleic acid from dead bacteria is equally amplifiable. Therefore, molecular assays are unsuitable for treatment monitoring and/or for infection control purposes.
We report an innovative approach to selectively amplify DNA derived from viable Mycobacterium tuberculosis in clinical specimens, which is useful for monitoring mycobacterial load in pulmonary TB patients during anti-TB treatment.
The protocol is based on pre-treatment of samples with propidium monoazide (PMA; Biotium Inc., Hayward, CA, USA), a chemical compound that can intercalate the DNA of non-viable (or membrane-damaged) organisms but is excluded from viable bacteria. After light activation, PMA binds covalently to the DNA, preventing its amplification by PCR [3]. After light exposure, unbound PMA is not able to interact further with DNA molecules.
The assay was first optimised using acid-fast bacilli (AFB)-negative sputum samples spiked with dead or live mycobacteria at different concentrations. In brief, live Mycobacterium fortuitum cells were added to N-acetyl-cysteine decontaminated sputum specimens negative for AFB by smear microscopy at a final concentration of 106 bacteria·mL−1. An aliquot of this laboratory-made sample was treated to heat kill the M. fortuitum cells. PMA stock solution was prepared and stored at -20°C and protected from light exposure, until use, as recommended by the manufacturers. PMA was added as a pre-treatment at a final concentration of 500 μM and incubated for 30 min at 4°C in the …