Rapid mycobacteria drug susceptibility testing using Gel Microdrop (GMD) Growth Assay and flow cytometry

• Published in: Journal of microbiological methods 2005-08, Vol.62 (2), p.181-197
• Main Authors: Akselband, Y., Cabral, C., Shapiro, D.S., McGrath, P.
• Format: Article
• Language: English
• Subjects: Antibiotics, Antitubercular - pharmacology; Bacterial diseases; Bacteriology; Biological and medical sciences; Drug Resistance, Multiple, Bacterial; Drug susceptibility to isoniazid (INH); Ethambutol (EMB); Ethambutol - pharmacology; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Gel Microdrop (GMD) Growth Assay; Gels; Human bacterial diseases; Humans; Infectious diseases; Isoniazid - pharmacology; Medical sciences; Microbial Sensitivity Tests - methods; Microbiology; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Microspheres; Mycobacterium - drug effects; Mycobacterium - growth & development; Mycobacterium avium; Mycobacterium avium - drug effects; Mycobacterium avium - growth & development; Mycobacterium bovis - drug effects; Mycobacterium bovis - growth & development; Mycobacterium tuberculosis; Mycobacterium tuberculosis - drug effects; Mycobacterium tuberculosis - growth & development; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Rifampin (RIF); Rifampin - pharmacology; Streptomycin (STR); Streptomycin - pharmacology; Tuberculosis and atypical mycobacterial infections
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2005.02.012

Control of multi-drug-resistant tuberculosis has been hampered by the lack of simple, rapid and sensitive methods for assessing bacterial growth and antimicrobial susceptibility. Due to the increasing incidence and high frequency of mutations, it is unlikely that culture methods will disappear in the foreseeable future. Therefore, the need to modernize methods for rapid detection of viable clinical isolates, at a minimum as a gold standard, will persist. Previously, we confirmed the feasibility of using the Gel Microdrop (GMD) Growth Assay for identifying sub-populations of resistant Mycobacteria by testing different laboratory strains. Briefly, this assay format relies on encapsulating single bacterium in agarose microspheres and identifying clonogenic growth using flow cytometry and fluorescent staining. In this study, we modified the GMD Growth Assay to make it suitable for clinical applications. We demonstrated the effectiveness and safety of this novel approach for detecting drug susceptibility in clinically relevant laboratory strains as well as clinical isolates of Mycobacterium tuberculosis. Correlation between results using the GMD Growth Assay format and results using two well characterized methods (Broth Microdilution MIC and BACTEC 460TB) was 87.5% and 90%, respectively. However, due to the inherent sensitivity of flow cytometry and the ability to detect small (