White-to-opaque switching is involved in the phospholipase B production of Candida dubliniensis on Price's egg yolk agar.

Measuring the production of Candida dubliniensis (C. dubliniensis) phospholipase B (PLase B) by the Price's method has long been considered to be unattainable because the levels of PLase produced are undetectable. In this study, C. dubliniensis, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis and C. tropicalis were shown to produce PLase B and form clear white zones around their colonies when peptone, a component of the original Price's egg yolk (OP) agar, is replaced with a yeast nitrogen base (YNB). This new medium is named modified Price's (MP) agar. Based on this finding, we propose a new modified Price's (NMP) agar containing 0.75% peptone and 0.25% YNB, which enabled measurement of PLase B production by C. dubliniensis and C. albicans with results consistent with those obtained for C. albicans grown on OP agar. We strongly believe that the MP and NMP agars are very useful for screening PLase B production by C. dubliniensis and non-albicans Candida spp. Moreover, the addition of several bioactive agents (the proteinase inhibitors pepstatin A and saquinavir, the calcineurin inhibitors cyclosporine A and tacrolimus, the cell-permeable cAMP analog dBcAMP, and the quorum-sensing molecule farnesol) to the OP agar enhanced PLase B production by C. dubliniensis. During the course of our study to clarify the reason why PLase B was not produced, we found that C. dubliniensis cells grown on OP agar undergo a white-to-opaque transition, which may explain why they showed minimal production of PLase B on this medium.

In vitro antifungal activity of luliconazole against clinical isolates from patients with dermatomycoses.

The in vitro antifungal activity of luliconazole, a novel topical imidazole, against pathogenic fungi implicated in dermatomycoses was studied. A total of 91 clinical isolates, consisting of 59 Trichophyton rubrum isolates, 26 T. mentagrophytes isolates, 1 Epidermophyton floccosum isolate, and 5 Candida albicans isolates were tested by the broth microdilution method, employing lanoconazole, terbinafine, and bifonazole as reference drugs. The minimum inhibitory concentrations (MICs) of luliconazole against T. rubrum and T. mentagrophytes were in the range of 0.00012-0.004 microg/ml and 0.00024-0.002 microg/ml, respectively. The MIC90 of luliconazole for these two species of dermatophytes was the same, at 0.001 microg/ml, and these values were 4 times, 30 times, and more than 1000 times lower than those of lanoconazole, terbinafine, and bifonazole, respectively. Similarly, the 1 isolate of E. floccosum tested was inhibited by luliconazole with an MIC of 0.001 microg/ml. Luliconazole also proved to be very potent against C. albicans (MIC range, 0.031-0.25 microg/ml), nearly on par, in terms of efficacy, with lanoconazole (0.063-0.25 microg/ml) and more potent than terbinafine (2->64 microg/ml) and bifonazole (0.5-4 microg/ml). These results showed that luliconazole was very potent in vitro against pathogenic fungi isolated from patients with dermatomycoses, and these findings emphasized the utility of luliconazole for the topical management of this condition.

A novel mycological analysis valuable for evaluating therapeutic efficacy of antimycotics against experimental dermatophytosis in guinea pigs.

Although antimycotic effects are mainly evaluated with regard to whether or not the fungi grow from a specimen obtained from the drug-treated skin, the potential for discrepancies in skin specimens in which the fungi are grown has not been evaluated, in the experimental tinea model. In this study, to evaluate the therapeutic effectiveness of antimycotic agents against fungal skin infection, a novel form of mycological assessment, which focuses on the size of colonies grown from skin specimens was examined and developed. When microconidia of Trichophyton mentagrophytes were inoculated onto a Sabouraud dextrose agar (SDA) plate and incubated at 27 degrees C for 5 days, a linear relationship was observed between the growth area of mycelia and the logarithm of the quantity of microconidia. This relationship between the growth area and the logarithm of the number of T. mentagrophytes microconidia did not change with the addition of skin homogenate and/or keratin powder. Next, the number of fungi in skin blocks attendant upon experimental, cutaneous infection in guinea pigs was evaluated and analyzed via a calibration curve, determined based on a microconidium suspension of T. mentagrophytes. Estimates of severity of dermatophytic infection in experimental animals were parallel to, but more reliable than, results obtained via the conventional mycological method (fungus-positive skin ratio of treated skin) in culture studies of infected dermal tissues. This new analytical method may also be applicable to the in vivo assessment of the therapeutic effect against dermatophytosis experimentally produced in guinea pigs.

A novel method using micropig stratum corneum in vitro for the evaluation of anti-Trichophyton mentagrophytes activity.

Antifungal susceptibility testing under conditions close to clinical status is expected to provide more helpful information than that obtained by a conventional microdilution method. For this purpose, we developed a novel method to evaluate anti-Trichophyton mentagrophytes activity of antifungal agents in vitro by using disks of micropig stratum corneum epidermis (SCE). Basal agar medium containing K2HPO4, MgSO4, CaCl2 and three kinds of antibiotics. Bifonazole (BFZ), lanoconazole (LCZ) or terbinafine (TBF) was added to the basal agar medium to give serially doubling dilutions ranging from 0.0006 to 10 microg/ml. Five-hundred-microl portions of the agar media thus prepared were solidified in wells of flat-bottomed plates. SCE disks (6 mm in diameter) were placed on surfaces of the agar medium and 10(4) conidia of T. mentagrophytes were inoculated on each SCE disk. There was very good correlation between the initial concentration of the antifungal agents added to the basal agar medium (microg/ml) and the concentration of the agents impregnated into the SCE disks (microg/g) (r2>0.99). The minimum inhibitory concentration (MIC) values of BFZ, LCZ and TBF were respectively 26-, 10- and 78-times higher than those measured by the standard microdilution method. From the correlation between the concentration of the agents in the basal medium and that in the SCE disks, the above MIC values corresponded to the concentrations in SCE disks (microg/g), 832.95 for BFZ, 1.42 for LCZ and 8.87 for TBF. This novel method of antidermatophytic susceptibility testing using SCE would be useful as an in vitro screening of proper antimycotics for topical treatment of dermatophytosis.

Development of a new medium useful for the recovery of dermatophytes from clinical specimens by minimizing the carryover effect of antifungal agents.

Two surface-active compounds, egg lecithin and polysorbate 80, usually used as the deactivators of various preservatives were tested whether they also counteract either or all of the three major topical antifungal drugs, bifonazole (BFZ), lanoconazole (LCZ) and terbinafine (TBF). Both egg lecithin and polysorbate 80, when added to culture media up to final concentrations of 1.0 and 0.7%, respectively, antagonized the anti-dermatophytic activity of the three drugs in a concentration-dependent manner. A greater extent of antagonistic action was exerted when the two deactivators combined at their maximal levels tested were added; MIC's of BFZ were increased more than 30-fold and those of LCZ and TBF more than 200-fold compared with the values obtained in the absence of the deactivators. Using the agar medium supplemented with the combined deactivators, culture studies were carried out with skin tissues specimens taken from guinea pigs whose feet were infected with dermatophytes and subsequently treated with 1% topical preparations of the three antifungal drugs. The experimental data from this animal study demonstrated that the combined deactivators-supplemented medium yielded increased numbers of fungi compared with the basal medium. It looks, therefore, likely that the fungal recovery on the former medium more correctly reflects to actual fungal burden in the infected lesions than the latter. All these results suggest that the combined deactivators-supplemented medium is more useful for mycological evaluation of therapeutic efficacy of imidazole and allylamine drugs against dermatophytoses in both preclinical and clinical studies.

Experimental tinea unguium model to assess topical antifungal agents using the infected human nail with dermatophyte in vitro.

Therapy for onychomycosis is difficult because a complete cure requires long-term treatment. Although strong systemic antifungal drugs are potent enough to achieve a cure, they often have side effects. Therefore, one approach is to develop a new topical antifungal drug to act directly on the nail tissue. In this study, we developed a new in vitro model for assessing antifungal activity using the human nail. An O-ring was fixed with silicon bond to the dorsal surface of the nail. An antifungal agent applied to the surface will penetrate the nail tissue. The ventral side of the nail was placed on an agar plate inoculated with conidia of Trichophyton mentagrophytes. Nail specimens were infected with the fungi from the agar, and a clear fungal colony formed on the agar surrounding the nail on the fifth day of cultivation. After 14 days, the fungal colony in the control groups was shown to be expanding over the entire nail. The fungal colony in the group treated with sodium pyrithione had disappeared. Although the in vitro antifungal activity of sodium pyrithione has poor potency among the agents used, it showed remarkable antifungal activity, as assessed by image analysis. This model enables one to evaluate the activity of a topical antifungal agent that has penetrated human nail tissue, and it may facilitate research on a topical agent for onychomycosis.