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Safe susceptibility testing of Mycobacterium tuberculosis by flow cytometry with the fluorescent nucleic acid stain SYTO 16

¹Department of Microbiology, Faculty of Medicine, University of Porto, Alameda Prof. Hernani Monteiro, 4200 Porto, Portugal ²IPATIMUP – Institute of Pathology and Molecular Immunology of the University of Porto, Portugal ³Department of Microbiology, Hospital de S. João, Alameda Prof. Hernani Monteiro, 4200 Porto, Portugal

Correspondence C. Pina-Vaz micteam{at}clix.pt

Received February 5, 2004
Accepted July 16, 2004

The time needed to obtain susceptibility results of Mycobacterium tuberculosis using classical methodologies is still too long, and flow cytometry is a promising technique in the setting of the clinical laboratory, giving fast results. A safe, reliable and rapid method to study susceptibility to streptomycin, isoniazide, rifampicin and ethambutol is described. Isolates of mycobacteria, grown for 72 h in the absence or presence of antimycobacterial drugs in the mycobacteria growth indicator tube (MGIT), were heat-killed, stained with SYTO 16 (a nucleic acid fluorescent stain that only penetrates cells with severe lesion of the membrane) and then analysed by flow cytometry. Sixteen strains with different susceptibility patterns were tested and an excellent correlation with the BACTEC MGIT 960 protocol for susceptibility was shown. In contrast to resistant strains, sensitive strains lose their cellular integrity after incubation with antimycobacterial drugs, allowing SYTO 16 to penetrate the cells. Comparing the intensity of fluorescence of Mycobacterium cells incubated with antimycobacterial drugs with that of drug-free cells, after staining with SYTO 16, it was possible to distinguish between sensitive, intermediate and resistant phenotypes. Other cytometric assays have been described for mycobacteria susceptibility testing but these have lower accuracy and safety. The described flow cytometric assay is a simple, fast, safe and accurate way to determine susceptibility of M. tuberculosis.