CONICET | Buscador de Institutos y Recursos Humanos

Introduction andObjectives: Tuberculosis (TB) is a major cause of human mortality with 9 millionnew cases and nearly two million deaths annually; approximately two billionpeople are infected with the causative agent, Mycobacterium tuberculosis (M.tb). The emergence ofresistant strains has become a serious public health problem worldwidecomplicating treatment and control of the disease. Traditional methods fordrug-susceptibility testing (DST) are slow and laborious, so there is a needfor new diagnostic methods that combine sensitivity, specificity, low cost andspeed. Reporter mycobacteriophages containing a fluorescent reporter gene are asimple mean of revealing the metabolic state of M.tb cells, and therefore their response to antibiotics. Until now,bacteria were detected after infection by fluorescence microscopy or by flowcytometry. Our aim was to evaluate the performance of Fluorophages directly insputum samples and to develop a simple, rapid and automated whole-cellscreening assay for DST of M. tb andalso applicable to HTS of new anti-TB drugs.Methods: We developed a mCherrybombɸ with higher sensitivity anda shorter time to detection of signal compared to the first generation ofFluorophages. We first tested the performance of the new mCherrybombΦ in negativeTB sputum samples spiked with M.tb mc26230using different processing protocols and infection conditions. After infection,cells were fixed with 4% PFA and detected using a fluorescence microscope,preserving the fluorescence and increasing biosafety. Then, positive TBclinical samples were used to evaluate the ability to identify M. tb and detect rifampicin resistance.We set up the conditions for infection of pure cultures in a multiwell formatin the presence of increasing concentrations of drugs and appearance offluorescence was monitored as a function of time using a fluorimeter. Theconcentration that inhibited 90% of fluorescence in the absence of drug was reportedas the minimal inhibitory concentration (MIC) for that drug. Results: We havedeveloped a second generation of Fluorophages with higher sensitivity and ashorter time to detection of signal, only 5 hs for M. tb. This new reporter phage could detect M.tb and determine rifampicin resistance directly in sputumsamples. Results were obtained between 24-120 hours from sputum collection.Using a fluorimeter, DST of M. tbcould be done from pure culture in 6 or 30 hs (when pre-incubation with thedrug was required). We found a good correlation between the MIC values obtainedwith this technique and the proportion method used as gold standard for TB. Conclusion: We were able to develop asimple and inexpensive assay for rapid detection and determination of rifampicinresistance of M.tb directly in sputumsamples. This new method could facilitate therapy and prevent the spread ofdrug-resistant strains in low resource settings. Finally, we had optimized theconditions for an automated phenotypic assay to test in a short timesusceptibility of pure cultures to different drugs used for TB treatment. Theimplementation of this standardized methodology will also contribute to thedevelopment of a rapid, easy and high sensitive method for HTS of new anti TBdrugs.