In vitro susceptibility testing for black grain eumycetoma causative agents

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.

Screening the pandemic response box identified benzimidazole carbamates, Olorofim and ravuconazole as promising drug candidates for the treatment of eumycetoma

“…Out of the nine azoles, fluconazole was only able to inhibit the growth of M. pseudomycetomatis, not M. mycetomatis and M. tropicana. Eumycetoma causative agents belonging to the order Pleosporales in general have higher MIC50 values towards the azoles than Madurella species [26], which was also demonstrated in this screening. Out of the nine azoles, only eberconazole was able to inhibit F. senegalensis growth in vitro, while none of the azoles was able to inhibit Me.…”

“…From our previous studies, we have determined that M. mycetomatis is most susceptible towards antifungals of the azole class [16,19,20,26]. It is therefore not surprising that out of the nine azoles included in the pandemic box, eight were able to inhibit M. mycetomatis growth below 25 μM.…”

Encapsulated eumycetoma caused by Biatriospora mackinnonii

“…Eumycetoma requires long-term medical therapy; however, the treatment is not entirely successful, showing low cure rates (26%) [ 2 ]. Low minimum inhibitory concentrations (MIC) for amphotericin B and azoles are often observed in B. mackinnonii ; moreover, MIC for azoles is three to four-fold dilutions higher than M. mycetomatis [ 12 ]; therefore, treatment duration often ranges between six months to three years, then surgical intervention is usually needed. Wide local surgical excision is the treatment of choice for non-responding eumycetoma and small, well-localized lesions, allowing better response to medical therapy [ 13 , 14 ].…”

Mycetoma, chromoblastomycosis and other deep fungal infections: diagnostic and treatment approach

“…Deep incisional biopsies are preferred to superficial samples, fine needle biopsies are also satisfactory [2]. Proper identification of the genus and species implicated is highly recommended, because not all pathogens involved are universally susceptible to azoles, the drugs of choice [5]. Molecular methods have emerged as the standard approaches to properly identify the fungal agent.…”