Fungal morphology after treatment with itraconazole as a single oral dose in experimental vaginal candidosis in rats.

The therapeutic effect of a single, oral dose of itraconazole was studied in rats inoculated intravaginally with Candida albicans and in which an established vaginal infection was present. We used light microscopy, transmission and scanning electron microscopy to document the structural alterations in the 3 days after treatment. The most important observations include the speed (within 24 hours) with which itraconazole inhibits the further penetration of the fungus into the vaginal squamous epithelium, the ability of the drug to reach and structurally alter intracellularly located fungal elements, and the prolonged drug effect of a single dose leading to complete eradication of the fungus from the vagina within 3 days.

The effects of saperconazole on the morphology of Candida albicans, Pityrosporum ovale and Trichophyton rubrum in vitro.

Fungal cultures were incubated for various periods of time with saperconazole at concentrations ranging from 10(-10) to 10(-5) M: Candida albicans (4 and 24 h), Pityrosporum ovale (2 and 7 days), and Trichophyton rubrum (1, 2, 3, 7 and 14 days). At the end of the incubation period, fungal morphology was compared with that of control cultures by transmission and scanning electron microscopy. In all three species, major ultrastructural changes were seen from concentrations of 10(-8) to 10(-7) M saperconazole onwards, depending on the species and time of incubation. The main changes were inhibition of hyphal outgrowth (C. albicans), abortive hyphal outgrowth (T. rubrum) and deposition of electron-dense vesicles in a thickened cell wall (C. albicans, T. rubrum). In P. ovale, a direct, necrotizing action was seen without concomitant wall changes.

Mode of action of itraconazole: morphological aspects.

The broad spectrum of antifungal activity of itraconazole is verified by morphologic criteria at the light and electron microscopical level. Yeast and fungal species examined are Candida albicans, Cryptococcus neoformans, Paracoccidioides brasiliensis, Sporothrix schenckii, Pityrosporum ovale, Trichophyton rubrum and Aspergillus fumigatus. Exposures of cultures of these yeasts and fungi to itraconazole results in dose- and time-dependent alterations which vary in nature and intensity from one species to another. The most striking gross morphological change is seen in the biphasic species and consists of the abolishment of morphogenic development of the blastospore into the hyphal forms (C. albicans and P. ovale) and of the hyphal into the yeast forms (P. brasiliensis). Furthermore, the outgrowth and development of inoculated A. fumigatus hyphae into sporulating vesicles is almost completely abolished. The above mentioned effects on morphogenesis are achieved in the 10(-10)-10(-7) M range. Except for P. ovale, the earliest ultrastructural changes after itraconazole consist of abnormalities at the plasma membrane, the cell wall and cytoplasmic vacuoles. They precede a marked increase in cell volume, defective cell division, abortive hyphal outgrowth and loss of cell viability. These changes are identical to those previously described after miconazole and ketoconazole treatment. However, when compared to the other available azole-derivatives sharing the same basic mechanisms of action, itraconazole displays an exceptionally strong activity against A. fumigatus which can be morphologically translated by a potent necrotizing action on hyphae and inhibition of vesicle formation and sporulation. The in vitro effects of itraconazole are supported by data obtained from microscopic examinations of samples derived from patients with experimental animals infected with various fungal organisms.

Structural degeneration of Aspergillus fumigatus after exposure to saperconazole.

Saperconazole is a newly synthesized triazole antifungal with potent activity against Aspergillus fumigatus. Exposure of spores inoculated into BHI agar medium to saperconazole doses as low as 35 ng ml-1, resulted in complete suppression of germination (hyphal outgrowth) when treatment started simultaneously with inoculation. Cultures which were grown for 24 or 48 h in the absence of drug and were then exposed to saperconazole showed a block in the development of hyphae, sporophores, vesicles, sterigmata and spores. Moreover, a substantial proportion of the pre-existent hyphae became necrotic during exposure to the drug. The latter was most obvious with the 70 ng ml-1 dose. Although treatment with lower doses yielded severely altered but non-necrotic cells, an abolishment of further outgrowth and differentiation was achieved.

Pityriasis versicolor and Pityrosporum ovale. Morphogenetic and ultrastructural considerations.

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) have been performed on skin material of patients with pityriasis versicolor who were orally treated with itraconazole. Before as well as after therapy, variously sized spherical structures were observed on the surface of the keratinocytes with SEM. TEM examination revealed Pityrosporum ovale, predominantly in its mycelial phase, inside keratinocytes. The spherules as observed by SEM appeared to be amorphous, lipid-like droplets originating from the inside of the keratinocytes. The cytoplasm of the keratinocytes was at least partly occupied by the same amorphous material. It is therefore suggested that P. ovale penetrates the keratinocyte where degradation of the normal keratinous content to amorphous material takes place. This newly formed lipidic substrate may be an essential nutritive factor. The lipidified state of the stratum corneum persisted for at least 3 weeks after eradication of the fungus by itraconazole. It is speculated that the presence of large quantities of this lipid-like material might be the cause of hypopigmentation because it may constitute an ultraviolet light block.

Degenerative changes in fungi after itraconazole treatment.

Changes in morphogenetic behavior and structural degeneration after exposure to itraconazole are illustrated in Candida albicans, Cryptococcus neoformans, Pityrosporum ovale, Paracoccidioides brasiliensis, Trichophyton rubrum, and Aspergillus fumigatus. With the exception of P. ovale, primary alterations are seen at the cell periphery and the cytoplasmic vacuoles in which lipid-like vesicles assemble. These changes are usually accompanied by a marked increase in cell volume, impaired cell division, or abortive hyphal outgrowth. The concentration of itraconazole necessary to induce irreversible structural degeneration (necrosis) depends greatly on the species used, the time of incubation, and the morphogenetic form in which the fungus is grown and varies from 10(-10) M (P. brasiliensis) to greater than 10(-6) M (C. albicans). Itraconazole achieves these effects either at a concentration comparable to that required for ketoconazole (C. albicans and C. neoformans); at concentrations 10- to 100-fold lower (P. ovale, T. rubrum, P. brasiliensis), or at concentrations 100-fold lower (A. fumigatus).

Itraconazole, a new triazole that is orally active in aspergillosis.

Itraconazole is a new orally active triazole derivative with broad-spectrum antifungal activity. This drug is effective in experimental aspergillosis and possesses in vitro activity against various species and strains of Aspergillus. Morphological destruction of inoculated hyphae and complete inhibition of hyphal outgrowth in culture is obtained from 0.07 micrograms ml-1 (10(-7)M) onward. These properties make itraconazole a likely candidate for clinical evaluation in disseminated aspergillosis.