In vitro susceptibility testing for black grain eumycetoma causative agents.

Eumycetoma is a neglected tropical implantation mycosis characterized by large subcutaneous swellings. Inside the infected tissue, the causative agents are found in grains. The most common causative agents form black grains and are sterile upon isolation. In vitro susceptibility assays were developed for eumycetoma causative agents. They were based on the Clinical and Laboratory Standards Institute M38A protocol and modified to enable the use of hyphae as a starting inoculum. To ease endpoint reading, viability dyes such as resazurin or XTT have been used. So far the in vitro susceptibility assays developed have mainly been used to establish if causative agents are inhibited in growth by various antifungal agents, but not for clinical decision making. For drug discovery, the assay proved useful in determining which compounds were able to prevent hyphal growth. However, a clear correlation between in vitro inhibition in terms of the half maximal inhibitory concentration or 50% minimum inhibitory concentration (MIC50) and therapeutic efficacy assayed in a novel model system in terms of Galleria mellonella larval survival was not found. For clinical decision making, a range of MICs were found for each antifungal agent. However, no clinical breakpoints have been established for any of the causative agents. For itraconazole, the MIC50 of most causative agents was below the attainable serum levels, which might indicate that they are susceptible. However, before in vitro susceptibility can be used in clinical decision making for mycetoma, a correlation between MIC and clinical outcome needs to be made.