Adequately designed preclinical and clinical studies are essential to evaluate newly developed antifungal drugs. This overview dealt primarily with problems to be solved to better assess the effectiveness of such drugs in systemic or topical use to treat deep mycoses and/or superficial mycoses.

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Preclinical experiments are extremely important for predicting the results of clinical trials in the development of novel antifungal agents. The methods of preclinical therapeutic evaluation of agents for treating dermatophytosis were discussed.
The agar plate dilution method has been conventionally used to determine in vitro antifungal activities. In 1995, the Committee Laboratory Standards, the Japanese Society for Medical Mycology proposed adoption of the micro broth dilution method used in testing drugs against deep-seated mycosis as the standard sensitivity testing method for yeast-like fungi. Although a final evaluation of the usefulness of this method remains to be made, its adoption in preclinical experiments in the near future is anticipated. Many problems, however, remain unresolved with regard to the standardization of sensitivity testing for filamentous fungi. In the micro broth dilution method, various end-point determination techniques have been used, such as mycelial ATP determination by bioluminescence measurement and spectrophotometric method based on the reduction of redox dye. The results of these techniques in sensitivity testing against dermatophytes were described.
Guinea pig infection models are commonly used to assess the in vivo therapeutic effects of agents against dermatophytosis. The usefulness of a tinea pedis model produced by infecting the foodplanta of guinea pigs with Trichophyton mentagrophytes, and the results of therapeutic effect and recurrence prevention assessments were presented. The assessment of recurrence prevention illustrated its importance as a model to study follow-up of drug effect in clinical trials.
Studies of drug retention in the epidermal corneum and persistence of drug effect are indispensable in the evaluation of antifungal agents against dermatophytosis. Methods for analyzing the pharmacokinetics of these drugs include affinity of the drug to keratin in vitro, prevention of symptom onset in animal models and cutaneous distribution of isotope-labelled drugs. Results of local pharmacokinetics in infected tissues together with antifungal activities of the agent allow a certain degree of prediction of therapeutic effectiveness in the skin.
Many antifungal agents that were evaluated favorably in these preclinical experiments have been confirmed to be effective in subsequent clinical trials.

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The developmental aim of antifungal agents for the treatment of deep seated mycoses is different from that of agents for superficial mycoses in two aspects. The formor must be active against fungal species which are common in deep seated infections not only, in vitro but also when administered systemically with their high bioavailability and safety. This means that their antifungal activities must be evaluable both in vitro and in vivo from an early stage of development. Proper in vitro evaluation of these agents is not easy however, because the results vary from experiment to experiment. A standard method of susceptivility testing were recently recommended for yeast, although not for molds. This method must be confirmed to make a good screening selection. Animal models are another way to determine the real activity of antifungals even at the screening stage, for in vitro tests often fail to provide this information. Pharmacokinetics, metabolism and safety tests are another important means of evaluation of antifungals. There are problems in estimating the results in human beings. The tests also take a lot of work and a long time to complete with some advantages to the existing agents.
Thus, it is easy to understand the difficulties involved in developing new fine antifungal agents. Clear standards including clinical trials are required for their development.

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Specimens which are positive on microscopy sometimes fail to grow on culture, which may make evaluation of the efficacy of an antifungal drug difficult. A new method for evaluating the viability of fungal elements using neutral red staining in skin scales indicates that this discrepancy is mainly explained by the fact that some of the fungal elements are non-viable. The antifungal effect of drugs can be evaluated by this staining in vitro. The viability of dermatophytes was evaluated by the method in skin scales taken from tinea patients treated with antifungal drugs. The results showed that discrepancy between the positive neutral red stain rate and the positive fungal element rate was remarkable at the end of follow-up. These findings indicate that the efficacy of antifungal drugs should not be evaluated by the existence of fungal elements in scales using KOH direct microscopic examination alone, but should be evaluated by their viability. In this respect, neutral red staining is a useful and easily applicable method for evaluation of the efficacy of antifungal drugs clinically.

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During the past several years substantial progress has been made in the development of new antifungal agents active against systemic fungal infections. Neverthless, despite the adverse effects associated with amphotericin B, it remains the golden standard therapy for most serious mycoses.
Five antifungal agents are commercially available in Japan for the treatment of systemic infections caused by fungi. These are amphotericin B, flucytosine, miconazole, fluconazole and itraconazole.
Flucytosine is a water-soluble analogue of cytosine that has a narrow spectrum of activity, which includes most strains of Cryptococcus neoformans and Candida sp. Most other fungi are resistant or have variable susceptibility. Flucytosine is usually administered in combination with amphotericin B to treat systemic mycoses, and in rare instances may be used alone. When flucytosine is used as a single agent, resistance often develops during therapy, especially in cryptococcal infections.
Miconazole was the first of the azoles used in the treatment of fungal infections in Japan, and reports of its efficacy in treating cryptococcosis or candidiasis have varied. Because of the development of other less toxic azoles, the utility of miconazole has diminished considerably.
Fluconazole was the first of the new triazoles and was approved for use in 1989. It has several therapeutic benefits and pharmacologic properties that differ from those of ketoconazole, and in addition, it is much less toxic than ketoconazole. It is highly water-soluble and can be administered intravenously to patients who are too ill to take medication orally.
Itraconazole is an orally active triazole derivative with broad-spectrum antifungal activity. A major difference from fluconazole is its greater activity against aspergillosis.
New antifungal agents are now under intense study. With their development and use in combination chemotherapy, we can expect further improvements in the treatment of opportunistic fungal infections.

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We isolated dermatophytes from normal looking feet of individuals who had bathed in four public baths in two Japanese hotels, one so-called “healthy land”, and one public bathhouse (sentou in Japanese). All volunteers were without lesions on their feet and revealed no dermatophytes by the foot-press method prior to bathing. Two male and two female volunteers were examined after bathing in each of the public baths using the same method. From eight male samples, a total of 230 colonies of dermatophytes were isolated; however, only seven colonies were detected from four out of eight female samples. Species of isolated dermatophytes included Trichophyton rubrum (61 colonies) and T. mentagrophytes (176 colonies). During and after this study, none of the volunteers developed any lesions on their feet.

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Fungal infections following bone marrow transplantation remain a major cause of morbidity and mortality. We reviewed 149 autopsies with bone marrow transplantation (BMT) in UCLA Medical Center for the period 1985-1994, focussing on the deep fungal infection.
Deep fungal infections were demonstrated in 47 (31.5%) of 149 patients with BMT. Causative fungi, commonly demonstrated in patients with BMT, were Aspergillus species and Candida species, and disseminated disease was found in approximately one-fourth of patients infected.
Examination of the incidence of causative fungi showed that candidiasis has been markedly decreasing since 1992, while aspergillosis has been increasing. It is suggested that the therapeutic difficulty of aspergillosis might have raised the mortality and incidence of invasive aspergillosis.

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We report a case of a 32-year-old healthy woman with superficial hyperkeratotic candidiasis of the palms, a rare type of superficial candidiasis. She visited our hospital because she developed scaly erythema with hyperkeratosis on both palms. Application of a topical steroid as well as plastic gloves were considered to be contributing factors. Candida albicans (sero-typeA) was isolated from scales of both palms. Direct examination using KOH on the first visit showed numerous hyphae but no spores that were not characteristic features of candidiasis, but on the second visit 2weeks later, the topical steroid had stopped showed characteristic features of candidiasis. After treatment with oral itraconazole, direct examination method and culture of the palms became negative and the lesions were almost cureed.

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Itraconazole (ITCZ), a triazole antifungal agent, was administered to 10 cases with proven or suspected pulmonary aspergillus infection, and its serum levels and tissue penetration were measured. In 9 cases with proven infection, the clinical efficacy was also evaluated.
Serum levels of ITCZ were well correlated with the daily dose, and a peak concentration of over 1, 300ng/ml was obtained in cases of 200-400mg daily 200-400mg for more than 8 days. In some cases the concentrations were lower than expected from the dosages, suggesting the difference in absorption and metabolism among individuals.
ITCZ showed good penetration into the lung tissue at about 2/3 of the serum level, however, level in the pleural effusion was relatively low. The penetration of ITCZ into sputum was also low at less than 10ng/g.
The clinical efficacy was evaluated considering all the other treatments and the efficacy rate was 77.8% (cure; 2, improved; 5, stable; 1 and died; 2). The results did not always reffect the serum levels of the drug. Even with long-term administration, no severe side effects were observed.
ITCZ is thus considered to be effective not only in the treatment of chronic infections and maintenance therapy for various types of pulmonary aspergillosis, but also in the treatment of some invasive-type infections with rapid achievement of high serum levels by relatively high loading dose at the start of the therapy.

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The combined effects of lysozyme and an azole antifungal, lanoconazole were investigated by microbroth dilution method against Candida albicans in vitro; growth of 7 of 8 C. albicans strains tested was inhibited for 14hr by 100∼200μg/ml of eggwhite lysozyme. IC 50 of the eight Candida strains, which is the concentration of lanoconazole causing 50% inhibition of growth, was estimated to be lower in the presence of 100μg/ml of lysozyme than in its absence. Using a typical strain of C. albicans, TIMM 1768, this combination was shown to be synergistic by analysis of the combined antimicrobial activity and was observed in Candida culture for as long as 72hr. Roles of this combination effect in anti-Candida therapy by lanoconazole were discussed.

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The effects of lanoconazole (LCZ) on the ultrastructure of hyphae of T. rubrum in a liquid medium. were investigated by ultrahigh-resolution low-voltage scanning electron microscopy (UHR-LVSEM) and transmission electron microscopy. SEM images revealed structural changes of the hyphae depending on the drug concentration: 1) when the minimum inhibitory concentration (MIC, 20ng/ml) of the drug was used, hyphae collapsed; 2) in 0.31-2.5ng/ml, there was curling and multibranching; 3) in 0.02-0.078ng/ml, there was exfoliation of the hyphal cell wall; and 4) phased exfoliation from outside to inside of the cell wall occurred concentration-dependently. The cell wall thickened and in these areas electron dense granules accumulated in accord with concentration. Thus LCZ, even at a low concentration of 1/1000 MIC, affects the ultrastructure of hyphae of T. rubrum especially through changes in the cell wall, and this leads to necrosis by degradation of the cytoplasmic structure.

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