Development and validation of in vitro microtiter-based viability assay incorporating resazurin for drug discovery and susceptibility testing against Madurella mycetomatis

• Main Author: Algaffar, Shereen S.O.
• Format: Other/Unknown Material
• Language: English
• Published: Morressier 2017
• Subjects: Sande; sami
• Online Access: https://openresearchlibrary.org/viewer/6b1cbd77-8bc4-434b-9a68-d21c5f165a2a; https://openresearchlibrary.org/ext/api/media/6b1cbd77-8bc4-434b-9a68-d21c5f165a2a/assets/external_content.pdf; https://doi.org/10.26226/morressier.5ac39996d462b8028d899fc6

Development and validation of in vitro microtiter-based viability assay incorporating resazurin for drug susceptibility testing and discovery of bioactive agents against Madurella mycetomatisShereen O. Abd Algaffar1, Wendy W. J. van de Sande2 and Sami A. Khalid1 1Faculty of Pharmacy, University of Science & Technology,2Erasmus MC, Department of Medical Microbiology and Infectious Diseases, Rotterdam, The Netherlands. ObjectivesAntifungals resistant and non-availability of a therapeutically effective drug against Madurella mycetomatis which is one of the most neglected tropical diseases prompted us to develop a rapid, reproducible, and inexpensive in vitro viability assay for drug susceptibility testing and as an indispensable tool in the drug discovery of novel bioactive agents against this disfiguring infectious disease.1 Resazurin (Alamar blue) assay offers a simple and affordable method especially in resource-limited settings along the u201cmycetoma endemic beltu201d and it is amenable to multiplexing applications with other assays, hence the incubation period is flexible, and the data can be collected using either fluorescence or absorbance detection.MethodsResazurin dye (0.15mg/ml) was employed as viability indicator in the present in vitro susceptibility testing of nine genetically identified clinical isolates of M. mycetomatis 2 using seven standard antifungal agents (Amphotericin B, Caspofungin, Fluconazole, Itraconazole, Ketoconazole, Voriconazole and Terbinafine). This assay was validated by comparison with XTT assay.3 The fungal inoculum used was ranging between 0.4 x 104 to 5 x 104 CFU/ml with 68-72% transmission. The mechanism of the resazurin method is based on the mitochondrial enzymes in the live cells that convert blue-coloured resazurin to a fluorescent pink-coloured resorufin proportionally to the number of metabolically active, viable cells present in a fungal population. The main differences between the two assays are revolving around that resazurin is added on the first day of incubation with distinctive colour on the 2-7th day of its incubation at 30-37 oC degree whereas XTT is added at the end of incubation period. ResultsA sharp, reproducible end-point was detected resulting from the conversion of resazurin to resorufin. Visual detection of MIC provided quantitatively identical absorbance measurements in resazurin assay. Comparison of the overall MIC values of resazurin assay with its counterpart XTT assay nearly provided the same MIC values in some isolates. The MIC of Amphotericin B ranges between 64-0.5ug/ml while the azoles except for Fluconazole, exhibited varying inhibitory effect on different isolates. Itraconazole and Posaconazole exhibited the lowest MIC value (>8-0.125ug/ml). Caspofungin and Terbinafine, however, showed weak inhibition on most of the isolates examined (MIC >256ug/ml).ConclusionA simple, reproducible resazurin-based in vitro assay was optimized for susceptibility testing of M. mycetomatis. The major advantages of the resazurin reduction assay are that it is relatively more sensitive than tetrazolium assays besides exhibiting the smallest variability across linear range, while much larger variations were observed for the MTT assays. References 1.tKhalid SA. 2nd ResNet NPND workshop on natural products against neglected diseases, November 25-28th, 2014, Rio de Janeiro, Brazil.2.tVan de Sande, WW. J et al. Journal of Clinical Microbiology, 43(9), 4349-56. 2005.3.tAhmed, A.O. et al. Antimicrobial Agents and Chemotherapy, 48 (7), 2742-46, 2004.