Comparison of Disc Diffusion, Etest, and a Modified CLSI Broth Microdilution Method for In Vitro Susceptibility Testing of Itraconazole, Posaconazole, and Voriconazole against Madurella mycetomatis

For many fungal infections, in vitro susceptibility testing is used to predict if an isolate is resistant or susceptible to the antifungal agent used to treat the infection. For Madurella mycetomatis, the main causative agent of mycetoma, in vitro susceptibility testing currently is not performed on a routine basis. The current in vitro susceptibility testing method is labor-intensive, and sonication must be done to generate a hyphal inoculum. For endpoint visualization, expensive viability dyes are needed. Here, we investigated if the currently used in vitro susceptibility method could be adapted to make it amendable for use in a routine setting which can be used in low-income countries, where mycetoma is endemic. First, we developed a methodology in which hyphal fragments can be generated without the need for sonication, by comparing different bead beating methodologies. Next, in vitro susceptibility was assessed using standard broth microdilution assays as well as disc diffusion, Etest, and VlPcheck methodologies. We demonstrate that after a hyphal suspension is generated by glass bead beating, disc diffusion, Etest, and VlPcheck can be used to determine susceptibility of Madurella mycetomatis to itraconazole, posaconazole, and voriconazole. The MICs found with Etest were comparable to those obtained with our modified CLSI-based broth microdilution in vitro susceptibility assay for itraconazole and posaconazole. Furthermore, we found an inverse relationship between the zones of inhibition and MICs obtained with the Etest and those obtained by the modified CLSI broth microdilution technique.