Establishing a method of inoculum preparation for susceptibility testing of Trichophyton rubrum and Trichophyton mentagrophytes

Abstract

A total of 92 clinical isolates of dermatophytes (52 of Trichophyton rubrum and 40 of Trichophyton mentagrophytes) were selected for testing with six antifungal drugs (terbinafine, griseofulvin, clotrimazole, miconazole, isoconazole, and fluconazole) and two pairs of drug combinations (ketoconazole-cyclopiroxolamine and itraconazole-cyclopiroxolamine). Two methods of inoculum preparation for susceptibility testing were evaluated that used (i) inocula consisting only of microconidia of dermatophytes filtered in Whatman filter model 40 and (ii) unfiltered inocula consisting of hyphae and microconidia. We followed the recommendations of approved document M38-A of CLSI (formerly NCCLS) with some adaptations, including an incubation period of 7 days and an incubation temperature of 28 degrees C. Reference strains of Candida parapsilosis, Candida krusei, Trichophyton rubrum, and Trichophyton mentagrophytes were included as quality-control strains. MICs were consistently higher (usually 1 to 2 dilutions for drugs tested individually) when nonfiltered inocula were tested (P < 0.01) except for terbinafine. Larger MICs were seen when testing drugs with nonfiltered inocula. The curves of drug interaction were used to analyze the reproducibility of the test, and it was shown that high levels of reproducibility were achieved using the methodology that included the filtration step. The standardization of methodologies is the first step to yield reliability of susceptibility testing and to proceed with clinical laboratory studies to correlate MICs with clinical outcomes.

FIG. 1.

Combinations of two pairs of drugs (ketoconazole-cyclopiroxolamine and itraconazole-cyclopiroxolamine) tested with Trichophyton rubrum (ATCC 40051) by use of different methods of inoculum preparation. Full lines represent data obtained with filtered inocula, and dashed lines represent data obtained with nonfiltered inocula. Irregular curves were obtained when unfiltered suspensions were tested. Results obtained with filtered inocula confirm the theoretical synergism for both pairs of drug combinations and exhibited high reproducibility.

FIG. 2.

Combinations of two pairs of drugs (ketoconazole-cyclopiroxolamine and itraconazole-cyclopiroxolamine) tested with Trichophyton mentagrophytes (ATCC 40004) by use of different methods of inoculum preparation. Full lines represent data obtained with filtered inocula, and dashed lines represent data obtained with nonfiltered inocula. Irregular curves were obtained when unfiltered suspensions were tested. Results obtained with filtered inocula confirm the theoretical synergism for both pairs of drug combinations and exhibited high reproducibility.