In this paper I reviewed basal studies of various recently developed types of antifugal agents, focussing mainly on their in vitro antimicrobial activity, therapeutic efficacy in animal models and/or mode of action. This information will be useful in the preclinical evaluation of a number of promising drugs. The groups of antifungal agents discussed here included: (i) thiocarbamates (tolciclate); (ii) 3-iodopropagyl derivatives (TJZ-100); (iii) triiodoallyl derivatives (CN-146), (iv) allylamines (naftifine and SF-86-327); (v) imidazole derivatives (tioconazole, bifonazole, sulconazole nitrate, oxiconazole nitrate, 710674-S, ketoconazole and miconazole) and triazole derivatives (terconazole, BAY n 7133 and ICI 153, 066); and (vi) wall-active antifungal antibiotics (echinocandins, aculeacins and papulacandins). Despite striking diversity in their chemical structure, all these groups of antifungals share the following biological properties: (1) they show a potent antifungal activity against a relatively wide range of pathogenic fungi, with the exception of thiocarbamates which are particularly active against dermatophytes but less or not active against most of other fungi; and (2) they are well tolerated by experimental animals and exhibit excellent therapeutic efficacy in animal models of superficial or deep-seated mycoses. It should also be stressed that there are several groups of antifungal agents which act on certain steps of biosynthetic pathways for essential cellular components specific for fungi, such as ergosterol as a major fungal membrane sterol and glucan or chitin as fungal wall polymers.

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We investigated the combined effect of amphotericin B (AMPH) and flucytosine (5-FC) with emphasis on their combined effect mechanism.
Investigations were focused on the susceptibility of four strains of Candida albicans isolated from clinical specimens to AMPH and in combination or alone 5-FC on yeast morphology agar. The cumulative mortality rate and histopathological findings in experimental Candida infections of mice treated with AMPH and/or 5-FC were studied, and determinations were made of intracellular concentrations of 5-FC of C. albicans in yeast nitrogen base with or without AMPH. We also determined the 5-FC levels in serum and various organs, and in urinary excretion of rats after administration of 5-FC alone or in combination of AMPH.
Synergistic effect of 5-FC and AMPH was found not only in in vitro but also in vivo experiments. No significant difference in pharmacokinetics of 5-FC by single and combined administration was observed and intracellular levels of 5-FC increased in parallel with the concentration of AMPH added.
These results indicated that the synergistic effect of 5-FC and AMPH in combination is caused by stimulation of the uptake of 5-FC into C. albicans cells by AMPH.

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Basic studies of antimycotics were reviewed mainly from the viewpoint of their mechanisms of action.
Antimycotics were examined as they affected growth inhibition against a cultured cell line of L1210, and were classified into 3 groups by cytotoxicity to L1210 cells. Such cytotoxicity was a good gauge of the toxicity of the antimycotic.
Combinations of antimycotics were studied in vitro to determine whether the activity of the agents in combination exceeded the sum of their activity when tested separately. Imidazole antimycotics were found to potentiate the antifungal effect of antimycotics with direct membrane damaging effect, especially for ionophores.
Emphasis was also laid on the combination effect of copiamycin and imidazole antimycotics, especially ketoconazole. Copiamycins are an ionophoretic antibiotic, and their remarkable enhanced efficacy was obtained in combination with ketoconazole. The combination effect was not only synergistic, but also strongly fungicidal.

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As an experimental approach to immunotherapy of mycotic infection, chitin and chitosan, with or without amphotericin B (AMPH) and flucytosine (5-FC) were assayed for their protective effect in mice challenged with a lethal amount of viable cells of Candida albicans NIH A-207 strain. Combination therapy with each pair of polysaccharide and chemotherapeutic agents gave a better result than that obtained by the corresponding single administration therapy.
To analyze the action mechanism of this combination effect, the peritoneal adherent cells (PAC) of mice treated with these polysaccharides were assayed for their killing effect on the viable cells of C. albicans in vitro. It was evident that PAC from chitin- or chitosan-treated mice showed a significantly increased killing effect in comparison with PAC of the corresponding untreated control, and that this effect was further enhanced by the coexistence of AMPH or 5-FC. Assay of myeloperoxidase activity of the peritoneal exudate cells (PEC) of mice treated with chitin or chitosan showed that a significant increase of this enzyme activity was observed in comparison with that of PEC of untreated mice.

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Recently many new antifingal agents have been developed for the treatment of dermatophytoses, mucocutaneous candidiasis and tinea versicolor. Among them, the agents most commonly used for the treatment of superficial dermatomycoses are the imidazole derivatives such as clotrimazole, miconazole, econazole, isoconazole, tioconazole and ketoconazole. Because these compounds have a broad spectrum of activity, they are effective in the treatment of all types of superficial dermatomycoses. Ketoconazole, especially, is the only orally administered agent among the imidazole derivatives. A double-blind comparison of ketoconazole and griseofulvin in the treatment of patients with tinea unguium was evaluated. At completion of therapy (twenty-four weeks), 39 (90.7%) of 43 patients treated with ketoconazole and 34 (85%) of 40 patients treated with griseofulvin were rated as markedly improved. No significant difference in therapeutic responses have been reported for either treatment group. A 13-year-old girl with chronic mucocutaneous candidiasis, who had failed to improve on conventional therapy, showed a notable clinical response to oral ketoconazole.

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The treatment of Japanese with sporotrichosis or black fungus infections is described. Cutaneous sporotrichosis is best treated by orally administered potassium iodide (KI) or local heat therapy. Ketoconazole and flucytosine (5-FC) were fairly effective in some patients with sporotrichosis. Thus, these two drugs may be useful for treating patients who fail to respond to KI or local heat therapy. Surgical excision is the treatment of choice in cases of black fungus infections with localized skin lesions. At present the most effective drug for the disease is 5-FC. In advanced cases with widespread lesions or internal involvement, a combined therapy using 5-FC and intravenously administered amphotericin B is recommended.
The mechanism of action of KI in sporotrichosis was also studied. After the administration of KI, no measurable amount of iodine was detected in the serum samples; however, serum levels of iodide reached 30-60ppm. The fungicidal activities of various reagents, alone or in combination, on Sporothrix schenckii were estimated in vitro. When peroxidase or ferrous iron was combined with iodide (1×10^-4M) and H₂O₂ (5×10^-5M), a fungicidal effect was complete 5min after incubation. Iodine (5×10^-6M) also exerted almost complete fungicidal effect at 5min in the presence of iodide. The combination of iodide and H₂O₂ showed a complete fungicidal effect at 120min. Preliminary experiment demonstrated that 2×10^-6M iodine was formed in the reaction mixture of 3×10^-4M iodide and 5×10^-5M H₂O₂. These results suggest that in addition to the peroxidase-iodide-H₂O₂ and iron-H₂O₂-iodide systems, iodine formed by the oxidation of iodide may exert an antifungal effect on S. schenckii.

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In the past few years, there has been an increase in the number of systemic fungal infections seen in clinical practice. The availability and widespread use of glucocorticoid and cytotoxic agents have resulted in many fungal infections in immunocompromised patients. In addition, patients receiving intravenous hyperalimentation, drug abusers, and patients with prosthetic valves are at increased risk of fungal infections.
Despite this increase in systemic fungal infections the development of effective antifungal agents has lagged well behind that of antibiotics.
Amphotericin B (AMPH) has been the most widely used and consistently effective antifungal agent over the past 30 years. It remains the treatment of choice for most of the deeply invasive or disseminated fungal diseases.
Flucytosine (5-FC) is used only infrequently as a single agent to treat serious disease, but the combination of AMPH and 5-FC is thought to have a synergistic effect against several different kinds of fungal infection.
The imidazoles currently available for clinical use include ketoconazole (KCZ) and miconazole (MCZ). The imidazoles are active in vitro against a wide range of dermatophytes, yeasts, dimorphic fungi, aspergilli, and actinomycetes. KCZ is water soluble and well absorbed from the gastrointestinal tract but should not be used to treat meningitis, since it does not penetrate well into cerebrospinal fluid. MCZ is available in intravenous preparation. There have been no randomized studies comparing MCZ with AMPH; it does appear to be less toxic than AMPH, but its efficacy in relation to AMPH has not been established. Therefore, it should be reserved for patients who cannot tolerate or have not responded to AMPH, or who are infected with an organism resistant to AMPH and sensitive to MCZ.
Finally, further studied on the mechanism of action of existing antifungal agents will lead to their more effective use, and more studies on the biology and pathophysiology of pathogenic fungi will provide information that is useful in the design of newer agents.

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The ratio of dermatophytoses patients to the total outpatients varied from 4 to 12% in various hospital reports. The ratio within each hospital, however, was in a relatively narrow range. The number of patients of tinea pedis gradually increased in each hospital with females recently numbering more than males. On the age distribution curve, female patients had a peak in the twenties, while males showed a plateau from the thirties to the fifties. Infantile cases of tinea pedis have also increased in past ten years. Patients with tinea unguium were distributed from youth to advanced age in males, but female patients were mainly of the younger generation and decreased in number with advanced age. The number of patients of tinea corporis was constant or slightly increased in many reports. On the other hand, tinea cruris has decreased in recent years and this decreasing tendency was found chiefly in young males. Tinea capitis and tinea barbae have also increased in recent years. The number of cutaneous candidiasis patients changed remarkably over the past 15 years. From 1971 to 1975 napkin rash-type candidiasis cases of infants extraordinarily increased in number, and then decreased rapidly a few years later. During the same five-year period intertriginous candidiasis also increased; although it subsequently decreased, it did not reach its former level and has slightly increased again in recent years. The incidence of candidial interdigital erosion, paronychia and onychia has not greatly changed over the past 15 years.

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Reported sporotrichosis, dematiaceous fungus infection and cryptococcosis cases in Japan were statistically and epidemiologically analysed.
1) Cutaneous sporotrichosis: As of December 31, 1983, 1, 396 cases of cutaneous sporotrichosis had been reported. Cumulative prevalence was calculated, 1.18 cases in 100, 000 population. 51.4% of the cases were of the fixed cutaneous type, 47.7% of the lymphocutaneous type and 0.5% of the disseminated type. It was of interest that massive fungal elements were detected in 11 cases, almost all of which were treated with steroid ointments.
2) Cutaneous dematiaceous fungus infections: Until December 31, 1983, 344 cases of cutaneous dematiaceous fungus infections had been reported. Cumulative prevalence was of 0.29 cases in 100, 000 population. 302 cases were diagnosed as chromomycosis (Fonsecaea pedrosoi, 264 cases; Phialophora verrucosa, 4 cases, Exophiala dermatitidis, 16 cases; Exophiala jeanselmei, 3 cases; others, 15 cases), 39 as subcutaneous phaeohyphomycosis or phaeomycotic cyst (Fonsecaea pedrosoi, 3 cases; Phialophora verrucosa, 3 cases; Exophiala jeanselmei, 25 cases; Dactylaria gallopava, one case, others, 7 cases) and 3 as tinea nigra palmaris (Exophiala werneckii, 3 cases).
3) Cutaneous cryptococcosis: As of June 31, 1984, 54 cases of cutaneous cryptococcosis were reported. Cumulative prevalence was obtained in 0.05 cases per 100, 000 population. 17 cases were classified as primary cutaneous, localized type, 7 as primary cutaneous, systemic type and 30 as secondary cutaneous, systemic type.

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We distributed a questionnaire to determine the number of cases of keratomycosis in Japan during one year and learned that, 176 cases had been diagnosed as keratomycosis during the year 1983. We indicated their distribution and discussed the method of diagnosis and treatment of keratomycosis in Japan.

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A total of 2, 913 mycosis cases up to 1981 as determined from questionnaire responses and the literature and checked for duplication were studied statistically and epidemiologically.
The patients comprised 1, 124 cases of asprgillosis, 824 cases of candidiasis, 663 cases of cryptococcosis, 131 cases of mucormycosis, 52 cases of nocardiasis and 119 cases of double or triple infected mycosis. These cases were classified into groups by year, sex, age and district for subsequent epidemiological survey, and were also checked for disease type, incidence of sequela, underlying disease, diagnostic method and prognosis.
From the response rate to a questionnaire sent out by the author, ratio of diseases reported and pathological findings reported on the autopsy records, etc., the number of patients with visceral mycosis in Japan was estimated.
Probable trends with the times in the types of mycosis were discussed based on the changes with generations. This information will throw some light on the present status of visceral mycosis in this country.

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The number of latent Mizumushi (Trichophytia pompholyciformis) patients in Japan was estimated from a questionnaire on the diseases sent to adults, including university students under twenty years of age. The amount of production and consumption of anti-fungal agents was also examined.
3, 076 people answered the questionnaire (1, 643, males and 1, 433 females). Respondents were divided into three groups according to their experience with Mizumushi: group 1, those having experienced the disease (37.8%); group 2, those possibly but not difinitely having experienced it (11.1%); and group 3, those without experience (52.5%).
Half of those in groups 1 and 2 answered that they were cured; therefore, 25% of all those who replied might still have Mizumushi. 12% of groups 1 and 2 had used nonprescription anti-fungal agents but half of them had stopped using them, so only 6% of groups 1 and 2 use nonprescription drugs for Mizumushi treatment at present.
From these replies, we estimated that the number of latent patients in Japan (between twenties and seventies) who use nonprescription drugs is about 4, 300, 000, while an examination of the amount of nonprescription drugs produced put the figure at about 3, 300, 000.
Actually, however latent patients should include people who do not use anti-fungal agents or who use other drugs. When these were added to the above numbers, we obtained 11, 000, 000-15, 000, 000 as the actual number of latent patients suffering from this disease.

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We isolated a black yeast strain from nodules on the left buttock of a 71-year-old male which were histologically confirmed as Bowen's disease. The isolate was seen histologically as hyphae in the upper layer of the crust of a Bowen's lesion, and was identified as Exophiala jeanselmei by slide culture and scanning electrom microscopic study.
To our knowledge, this is the first report of a saprophytic growth of E. jeanselmei on a pre-existing skin lesion.
We consider that this case indicates an intermediate step between saprophytic growth and infection.

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A case of primary cutaneous aspergillosis caused by Aspergillus flavus and A. niger in a five and a half month old baby is presented. The lesions appeared following constant contact wear of orthopedic shoes and consisted of vesicles and pustules which are indistinguishable from those of tinea or candidiasis of the sole. The patient was successfully treated with topical application of isoconazole nitrate (Adestan cream^®) twice daily for three weeks.

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Alterations in lipid composition of purified plasma membranes from Candida albicans were examined during the yeast to hyphal transformation. The levels of both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) increased by 5 and 10%, respectively three hours after transfer, while phosphatidylserine (PS) and phosphatidic acid (PA) decreased by 10% respectively. Marked changes were also observed to occur in the fatty acid composition of phospholipids three hours after transfer. There was a great increase in linoleic acid with a compensatory decrease in oleic acid in PC, PE and PS, suggesting enhanced desaturation activity. Such replacement of monoene by diene led to the high unsaturation index associated with the yeast to hyphal transformation. The fatty acid composition of phosphatidylinositol showed a striking decrease in palmitoleic acid with a concomitant increase in palmitic acid. These results suggest that modification of plasma membrane lipid composition might be associated with the morphological conversion from yeast to hyphae in C. albicans.

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The first case of bovine protothecosis in Japan was described. The diseased cow showed clinical mastitis in the right front and left hind quarters with swelling, accompanied by lanquidness, poor appetite and mild fever. Prototheca zopfii was repeatedly isolated from milk specimens of the right front and left hind quarters during a 7 month period.
These isolates of P. zopfii grew well on Sabouraud's dextrose agar and their colonies were yeast-like. The multiplication was autospore formation. In an assimilation test, the isolates assimilated glucose, glycerol and ethanol, but did not assimilate sucrose, trehalose, n-butanol or n-propanol. Moreover, using the API 20C system, the isolates assimilated glucose and glycerol as well as reference strains of P. zopfii. The isolates showed low sensitivity to streptomycin, kanamycin, amphotericin B and nystatin. In a gel immunodiffusion test using a culture filtrate antigen of the isolates from the disease cow's milk, one clear band was demonstrated for sera of the diseased cow.
In experimental infection of mice using P. zopfii strains isolated from bovine mastitis, none of the mice died by intraperitoneal, intravenous or intratesticular injection. However, histopathological examination of sections by periodic-acid Schiff demonstrated that P. zopfii had infected the testicles of predonisolone-treated and non-treated mice and the liver of predonisolone-treated mice.

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The effects of nikkomycin (N. K.) on chitin synthetase of Candida albicans were studied and compared with polyoxin D and amphotericin B. N. K. was found to inhibit the synthetase activity. Its inhibitory action was much stronger than that of polyoxin D. Ki values were about 0.12μM for N. K. and 1.52μM for polyoxin D. The kinetic parameters indicate that the mode of action of N. K. appears to be very similar to that of polyoxin D. The results provide evidence that N. K. is a powerful competitive inhibitor of incorporation of UDP-N-acetyl-D-glucosamine into chitin in the cell wall.

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It is knownhat a spontaneous increase in IgE antibody is seen in the early stage of Kawasaki disease. Based on previous experiments of Candida extract-induced arteritis in mice, an investigation of the relationship between IgE antibody and the incidence of arteritis was carried out using the PCA method. It was confirmed that IgE antibody (PCA titer 1) was produced in response to two round immunization with alkali-extracted Candida antigen. It was also shown that the production of IgE antibody increased when Candida antigen was injected together with Bordetella pertussis vaccine. In this case, mice in a group which had B. pertussis vaccine in a first round immunization with Candida antigen showed higher IgE antibody titer than those in a group which had B. pertussis vaccine in a second round immunization. In each of three groups, the incidence of arteritis was 40%, 87% and 55% respecitvely. However, the mice in a group which only one round injection of a mixture of Candida antigen and B. pertussis vaccine showed neither PCA reaction nor incidence of arteritis. This result suggests that IgE antibody may be related to the incidence of arteritis.
The time course of IgE antibody production was also studied. The IgE antibody (PCA titer 20) appeared on the third day after the second round injection and reached a peak (PCA titer 80) on the 10th day, then decreased on the 24th day (PCA titer 1). It was also shown that the production of IgE antibody in this experiment resembled that in Kawasaki disease.

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Viable and heat-killed yeast form cells of Candida albicans (10⁸/ml, 0.1ml) were respectively inoculated in the skin of guinea pig flank. Follow-up and histopathological study of the skin biopsies showed the following. 1) Neutrophil chemotaxis was observed at the inoculation sites both of viable and heat-killed cell, but was more remarkable at the former. 2) Germ tube formation of viable cells occurred within a few hours after inoculation. Quantitatively, the germination was inversely related to the neutrophil infiltration. Degenerated and/or phagocytized fungal elements were seen in the nest of inflammatory cells. 3) A large number of viable cells in the dermis was characteristically surrounded by a two-layered wall of inflammatory cells. Thereafter, actively proliferating epithelium engulfed and continuously excluded them from the dermis to the exterior. This mechanism is explained as the phenomenon of transepithelial elimination. A few organisms were seen in the cytoplasm of histiocytes and giant cells outside the abscess. 4) Heat-killed cells were phagocytized and digested in the cytoplasm of histiocytes and giant cells without epithelial response.
These suggested that the development of deep dermal candidiasis may be inhibited by a defense system consisting of the inflammatory cell infiltration and proliferating epithelium in response to viable C. albicans. Furthermore, anastomosing follicular epithelium deserves a special emphasis of the transepithelial elimination.

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A 45-year-old woman occasionally had fever of unknown origin, headache, shift to the left of leukocytes, increase of blood sedimentation rate, positive CRP test and hypergammaglobulinemia from 5 years ago. She had high fever and marked increase of blood sedimentation rate one year ago and chest X-ray examination revealed an infiltrating lesion, approximately 1cm in diameter, in the upper field of the left lung. Under the diagnosis of pulmonary tuberculosis, antituberculous drugs (combination of isonicotinic acid hydrazide, ethambutol, streptomycin and rifampicin) with antibiotics were administered, but the lesion of the lung remained unchanged. Since bronchography disclosed three spindle-shaped bronchiectatic cavities, a lobectomy of upper lobe of the left lung replaced the antituberculous drug therapy.
Pathological examination of the resected lung tissue revealed that the cavitary lesions consisted of localized bronchiectasia containing Aspergillus hyphae in the lumen and associated with chronic inflammation surrounding fungal elements. In addition, some of their peripheral bronchioles were also filled with Aspergillus hyphae. However, no tuberculous lesion and bacilli were observed in the specimens.
It is considered that these findings may indicate primary broncho-pulmonary aspergillosis and a process to develop aspergilloma without antecedent cavity due to primary disease.

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