Antimycotic susceptibility testing of dermatophytes in microcultures with a standardized fragmented mycelial inoculum

Abstract

Standardization of a fragmented dermatophyte mycelial inoculum free of conidia for use in a broth microculture antimycotic susceptibility testing system is described. Ten clinical dermatophyte isolates were grown in submerged broth cultures at 35°C. The mycelia were harvested before conidia appeared and were fragmented to 10 to 50-,um segments with a Broeck ground-glass tissue grinder. The density of the fragmented mycelium preparation was found to be adjustable on the basis of its spectrophotometric density expressed as absorbance measured at 450 nm. The minimal absorbance density that produced a 100% inoculation efficiency was determined for each isolate, and from these data an absorbance density of 0.600 was selected for inoculation of microcultures used in antimycotic susceptibility testing. The 0.600-absorbance inoculum of each dermatophyte isolate was tested for its capacity to successfully inoculate antimycotic agentcontaining microcultures and generate minimal inhibitory concentrations of griseofulvin, clotrimazole, miconazole, and ketoconazole. The effect of the length of incubation on the minimal inhibitory concentrations was determined. It was concluded that the 0.600-absorbance density fragmented mycelial inoculum assured the rapid and uniform inoculation of the broth microculture antimycotic susceptibility system.

Collaborative Evaluation of Optimal Antifungal Susceptibility Testing Conditions for Dermatophytes

“…Similar results were obtained in a multicenter study performed by Espinel-Ingroff et al (8), who, testing filamentous fungi, found the lowest intra-and interlaboratory agreement for ITC (59 to 79% and 59 to 91%, respectively). Although several studies have been published on the in vitro susceptibility of the three drugs tested here (15,17,18,24,27), it is difficult to compare results due to variability in the different methods and conditions under which they were performed. We observed in general that TRB was more active than CLT and ITC.…”

“…Here, we have used two inocula measured spectrophotometrically, which allowed us to obtain suspensions with no particularly significant dispersion in the number of particles. (3,15,24), but their results have showed great variability. This is likely due to the lack of standardization of different parameters that can influence MICs determination, such as inoculum (14), length and temperature of incubation (7,20,21), and endpoint criteria (9).…”

“…In recent years, some authors, possibly encouraged by the development of the above-mentioned reference method, have published various articles wherein several species of dermatophytes have been tested (11,27,28,34). In these works, different adaptations or modifications of the NCCLS methods have been assayed, although other techniques have also been used (3,12,15). The results obtained have been clearly contradictory in some aspects, which makes evident the need for standardization and the development of reference methods.…”

Results of German multicenter study of antimicrobial susceptibilities of Trichophyton rubrum and Trichophyton mentagrophytes strains causing tinea unguium. German Collaborative Dermatophyte Drug Susceptibility Study Group

“…Itraconazole and terbinafine activities were assessed at concentrations of 2, 1, 0.5, 10 , and 10 Ϫ7 g/ml. The microdilution test described by Granade and Artis was used (5). The test is essentially a broth dilution test in microtiter plates.…”

Synthesis and antifungal activity of some 2-benzothiazolylthioacetyl amino acid and peptide derivatives

“…28 The agar diffusion method (well technique) was employed to determine the approximate minimal inhibitory concentration (MIC) values, the concentration considered inhibitory if minimal inhibition was found around the well.…”