Introduction Eumycetoma is a subcutaneous mutilating disease that can be caused by many different fungi. Current treatment consists of prolonged itraconazole administration in combination with surgery. In many centres, due to their slow growth rate, the treatment for eumycetoma is often started before the causative agent is identified. This harbours the risk that the causative fungus is not susceptible to the given empirical therapy. In the open‐source drug program MycetOS, ravuconazole and luliconazole were promising antifungal agents that were able to inhibit the growth of Madurella mycetomatis, the most common causative agent of mycetoma. However, it is currently not known whether these drugs inhibit the growth of other eumycetoma causative agents. Materials and methods Here, we determined the in vitro activity of luliconazole, lanoconazole and ravuconazole against commonly encountered eumycetoma causative agents. MICs were determined for lanoconazole, luliconazole and ravuconazole against 37 fungal isolates which included Madurella species, Falciformispora senegalensis, Medicopsis romeroi and Trematosphaeria grisea and compared to those of itraconazole. Results Ravuconazole, luliconazole and lanoconazole showed high activity against all eumycetoma causative agents tested with median minimal inhibitory concentrations (MICs) ranging from 0.008–2 µg/ml, 0.001–0.064 µg/ml and 0.001–0.064 µg/ml, respectively. Even Ma. fahalii and Me. romeroi, which are not inhibited in growth by itraconazole at a concentration of 4 µg/ml, were inhibited by these azoles. Conclusion The commonly encountered eumycetoma causative agents are inhibited by lanoconazole, luliconazole and ravuconazole. These drugs are promising candidates for further evaluation as potential treatment for eumycetoma.

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 fungal 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, E-testing and VIPcheck™ methodologies. We demonstrate that after a hyphal suspension is generated by glass bead beating, disc diffusion, E-testing and VIPcheck™ can be used to determine susceptibility towards itraconazole, posaconazole and voriconazole of Madurella mycetomatis . The MICs found with the E-test 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 zone of inhibition and MIC obtained with E-test and the modified CLSI broth microdilution technique.