Multilaboratory Evaluation of In Vitro Antifungal Susceptibility Testing of Dermatophytes for ME1111

Abstract

h ME1111 is a novel small molecule antifungal agent under development for the topical treatment of onychomycosis. Standardization of the susceptibility testing method for this candidate antifungal is needed. Toward this end, 8 independent laboratories determined the interlaboratory reproducibility of ME1111 susceptibility testing. In addition, we subsequently identified 2 strains as quality control (QC) isolates for the method. In the reproducibility study, 5 blinded clinical strains each of Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton floccosum were tested, while the QC study tested 6 blinded T. rubrum or T. mentagrophytes ATCC strains. Testing was performed in frozen microtiter panels according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 methodology. In the reproducibility study, 9 of 15 clinical strains showed interlaboratory agreement of >90% at the 80% inhibition endpoint, with a range of agreement of 76.2% to 100%. In the QC study, 4 of the 6 ATCC strains showed interlaboratory agreement of >90%. ME1111 demonstrated excellent interlaboratory agreement when tested against dermatophytes. Based on this data, the CLSI Subcommittee on Antifungal Susceptibility Tests approved the susceptibility testing of ME1111 against dermatophytes according to M38-A2 methodology, which stipulates RPMI 1640 as the test medium, an inoculum size of 1 to 3 ؋ 10 3 CFU/ml, and an incubation time and temperature of 96 h at 35°C. The MIC endpoint should be 80% inhibition compared with the growth control. T. rubrum ATCC MYA-4438 and T. mentagrophytes ATCC 28185 were selected as QC isolates, with an acceptable range of 0.12 to 1 g/ml for the two strains. Currently available topical agents for the treatment of onychomycosis have low efficacy, and thus, there is a continued need for the development of new topical antifungals (1, 2). ME1111 showed potent antifungal activity against Trichophyton rubrum and Trichophyton mentagrophytes (the major etiologic agents of onychomycosis) strains isolated in Japan and reference fungal strains with an MIC range of 0.12 to 0.5 mg/liter and with an MIC 50 and an MIC 90 of 0.5 mg/liter for both (3). ME1111 is a novel antifungal whose small molecular weight enhances its ability to penetrate the nail plate to reach the nail bed where the infection resides. ME1111 inhibits succinate dehydrogenase (complex II) (4), and the fungicidal activity of ME1111 seems to be due to its inhibition of this enzyme, which leads to the blockade of ATP production.The standardized method for the susceptibility testing of dermatophytes was added to the Clinical and Laboratory Standards Institute (CLSI) approved standard M38-A2 following an interlaboratory study involving ciclopirox, fluconazole, griseofulvin, itraconazole, posaconazole, terbinafine, and voriconazole (5). However, this standard does not specifically reference ME1111.Therefore, in order for ME1111 to be included in this standard, an interlaboratory study was needed to evaluate the reproducibility of the MICs of ME111...

Antifungal susceptibility testing of dermatophytes: Development and evaluation of an optimised broth microdilution method

“…Finally, we investigated whether to read the MIC values on the 4th or 7th day. We mostly observed enough growth after 4 days, which is in line with other studies 10,20,27 . After 7 days, generally equal or higher MICs were observed.…”

“…Similar as for the temperature, the inoculum concentration is also a matter of debate (Table 1). While in some studies 18,20,21,27 an inoculum density of 10 3 CFU/mL was used, other studies 22,24‐26,28 showed reproducible results with a concentration of 10 4 CFU/mL. The CLSI reference method for broth dilution antifungal susceptibility testing of filamentous fungi, specifically recommends an inoculum of 1–3 × 10 3 CFU/mL for dermatophytes, while for nondermatophyte moulds 0.4–5 × 10 4 CFU/mL is recommended 9 .…”

Antifungal drug susceptibility testing of dermatophytes: Laboratory findings to clinical implications

“…[45] The studies reported that micro broth dilution method requires incubation at 35°C and should be read at the end of 72-96 hour. [484950] Though the incubation period of 3-4 days is generally accepted to read the results, the time duration may exceed for a slow growing dermatophyte. Hence, final reading should be read on the basis of presence of growth in the control well.…”

Morphological Effect of the New Antifungal Agent ME1111 on Hyphal Growth of Trichophyton mentagrophytes, Determined by Scanning and Transmission Electron Microscopy

“…It is highly active in vitro and in vivo against Trichophyton rubrum and Trichophyton mentagrophytes and shows excellent human nail permeability (1)(2)(3)(4). A previous study on its mechanism of action revealed that the molecular target of ME1111 was succinate dehydrogenase (complex II) in the mitochondrial electron transport system (5).…”