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In vitro susceptibility testing for black grain eumycetoma causative agents
1Erasmus Medical Center, University Medical Center Rotterdam, Department of Medical Microbiology and Infectious Diseases, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.
Abstract:
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.
Insights
Developing in vitro susceptibility assays for eumycetoma agents is crucial for antifungal drug discovery. However, current methods lack direct correlation to clinical outcomes, necessitating further research for reliable decision-making.
Area of Science:
- Medical Mycology
- Tropical Diseases
- Antimicrobial Resistance
Background:
- Eumycetoma is a neglected tropical mycosis causing subcutaneous swellings, with causative agents found in grains within infected tissue.
- The most common eumycetoma agents produce black grains and are sterile in culture, complicating diagnosis and treatment.
- Standardized in vitro susceptibility assays are needed to guide antifungal therapy for eumycetoma.
Purpose of the Study:
- To evaluate the utility of in vitro susceptibility assays for eumycetoma causative agents in drug discovery and clinical decision-making.
- To assess the correlation between in vitro antifungal activity and therapeutic efficacy in a model system.
- To determine the feasibility of establishing clinical breakpoints for eumycetoma treatment.
Main Methods:
- In vitro susceptibility assays were developed based on the Clinical and Laboratory Standards Institute M38A protocol, modified for hyphal inoculum.
- Viability dyes like resazurin or XTT were employed to facilitate endpoint determination.
- Antifungal compound efficacy was tested against eumycetoma agents, and results were compared with Galleria mellonella larval survival models.
Main Results:
- The assays successfully determined antifungal inhibition and were useful for drug discovery by identifying compounds preventing hyphal growth.
- A clear correlation between in vitro measures (MIC50) and therapeutic efficacy in the Galleria mellonella model was not established.
- While some agents showed MIC50 values below attainable serum levels (e.g., itraconazole), clinical breakpoints are yet to be defined.
Conclusions:
- In vitro susceptibility assays are valuable for antifungal drug discovery against eumycetoma agents.
- Further research is required to correlate in vitro results with clinical outcomes before these assays can guide mycetoma treatment decisions.
- Establishing clinical breakpoints is essential for the effective use of in vitro susceptibility data in managing eumycetoma.
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