Madurella mycetomatis (M.mycetomatis) is the major causative agent of eumycetoma. Currently, susceptibility testing for M. mycetomatis is not routinely performed, due to the labour-intensive method, the long incubation times and use of expensive dyes for endpoint visualization.
Here we determined if the Sensititre yeastOne YO10 Alamar blue assay can be used to determine the in vitro susceptibility of M. mycetomatis towards several antifungal agents. To assess if pyomelanin formation interfered with the read-out systems, M.mycetomatis isolates with and without pyomelanin formation were compared.
The susceptibilities of 10 clinical isolates of M. mycetomatis were determined with Sensititre YeastOne YO10 Alamar blue assay following the manufacturer’s instruction. The Minimum Inhibitory Concentrations, (MICs) were determined by visual reading, SWIN reading and Spectrophotometric reading and the levels of agreement between various read-out methods were compared.
The lowest MICs were observed with the triazoles itraconazole, posaconazole and voriconazole with MIC50s of 0.03-0.06 g/ml for all readout methods. High MICs were observed for echinocandins and 5-flucytosine for all readout methods with MIC50s all >8 g/ml and 64 g/ml respectively, while variable for amphotericin B. A high interexperimental agreement was observed between visual and SWIN readings. The levels of agreement were between 30-100% and 70-100% for identical MICs and MICs within 1 dilution step respectively. Equally, the agreement between visual and spectrophotometric readings was considerably good ranging between 0-90% and 30-100% for identical MICs and MICs within 1 dilution step difference respectively. Furthermore, the agreement between SWIN and absorbance readings was higher compared to visual and absorbance. This agreement range was within 20-90% and 50-100% for identical MICs and MICs within 1 dilution step difference respectively. Pyomelanin secretion had an influence on the expected colour change but no effects on the MICs determination for all antifungals tested.

Comparison of three read-out methods for Sensititre YeastOne Alamar blue assay for determining Minimum Inhibitory Concentrations