The defect in host defense that makes the diabetic ketoacidotic (DKA) patient susceptible to mucormycosis has not been identified. Sera from 10 DKA patients and three normal volunteers were tested for their capacity to support the in vitro growth of a common etiologic agent of mucormycosis, Rhizopus oryzae. After equilibration with room air none of the normal or DKA sera, each of which was now extremely alkaline, supported growth of R. oryzae. When the sera were placed in a CO2 atmosphere that permitted simulation of the in vivo clinical pH (normal 7.40 and DKA 7.3-6.6), four of seven DKA sera supported profuse fungal growth. No growth occurred in normal serum. The three DKA sera that did not support fungal growth at pH less than or equal to 7.3 contained less iron (x = 13 micrograms/dl) than the four sera that supported profuse fungal growth (x = 69 micrograms/dl). Increasing the iron content of iron-poor DKA serum that did not support R. oryzae growth allowed profuse growth at acidotic conditions but not at pH greater than or equal to 7.4. Simulated acidotic conditions (pH 7.3-6.6) also decreased the iron-binding capacity of normal serum stepwise from 266 micrograms/dl to 0. Our data indicate that acidosis temporarily disrupts the capacity of transferrin to bind iron and suggest that this alteration abolishes an important host defense mechanism that permits growth of R. oryzae.

Delivery of ketoconazole to human stratum corneum was studied. Thirteen healthy volunteers, three patients with chronic fungal disease and one patient with palmar-plantar hyperhidrosis were given 400 mg of ketoconazole daily for various lengths of time. The ketoconazole content of palmar stratum corneum, eccrine sweat, sebum, and serum was measured by high-pressure liquid chromatography (sensitivity, 0.005 to 0.010 microgram/ml). Palmar stratum corneum obtained after 7 and 14 days of daily administration contained up to 14 micrograms of ketoconazole per g. Ketoconazole was not found in sebum after 7 or 14 days of daily ingestion of the antimycotic agent. Sebum from three patients with chronic fungal infection treated for greater than 9 months contained ketoconazole (means, 4.7 micrograms/g). Thermogenic whole body eccrine sweat contained a mean of 0.059 microgram/ml on day 7 and 0.084 microgram/ml on day 14 of daily administration. Ketoconazole appeared in thermogenic whole body eccrine sweat and palmar hyperhidrotic sweat within 1 h after a single oral dose. Partition studies of ketoconazole containing eccrine sweat demonstrated a 10-fold greater concentration in the sediment phase (desquamated keratinocytes) compared with the clear supernatant phase. In vitro studies with [3H]ketoconazole-supplemented supernatant sweat revealed preferential binding to stratum corneum, hair, and nails and its partitioning to lipid-rich sebum. We conclude that eccrine sweat rapidly transports ketoconazole across the blood-skin barrier, where it may bind or partition to keratinocytes and surface lipids.

It has been suggested that siderophores may function as virulence factors. There have been few studies on production of siderophores by opportunistic and pathogenic fungi. We examined siderophore production by Absidia corymbifera, Aspergillus niger, Rhizopus arrhizus, Rhizopus oryzae, Blastomyces dermatitidis, Histoplasma capsulatum, Sporothrix schenickii, Candida albicans, and Trichophyton mentagrophytes. Fungi were cultured at 37 and 27°C in a chemically defined low-iron media (0.2 ,uM Fe). Culture supematants were assayed for siderophores by two nonspecific methods [FeC13 and Fe(CI04)3] and three chemically specific assays (catechol, 2,3-dihydroxybenzoate, and hydroxamate). All fungi secreted siderophores. Only siderophores of the hydroxamate type were found. More siderophore was produced at 27°C than at 37°C. The present study adds eight fungi to the list of known siderophore producers and confirms siderophore production by H. capsulatum.

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.

Monitoring of filamentous fungal growth by spectrophotometry is generally considered not feasible. This report describes the monitoring of growth of the filamentous fungi Trichophyton mentagrophytes, Rhizopus oryzae, and Sporothrix schenckii in broth by two new spectrophotometric methods and by 14C incorporation from [U-_4C]glucose. Microcultures (200 ,lh) were prepared in 96-well, flat-bottom microtiter trays, and macrocultures (4 ml) were prepared in glass vials proportionally scaled up from microcultures. Mycelium accumulation in microcultures was measured without terminating the cultures by in situ microspectrophotometry. Accumulation in macrocultures was monitored by uniformly fragmenting the mycelium with a Broeck tissue grinder and by measuring absorbance density in plastic cuvettes with a dual-beam spectrophotometer. Absorbance measurements were found to increase linearly with mycelial weight. In situ absorbance correlated with absorbance density of fragmented mycelium, indicating that both methods monitored growth equivalently. Both defined lag-, exponential-, and stationary-growth phases. Increases in 14C incorporation, absorbance, and mycelial dry weight were kinetically identical for macrocultures and microcultures of T. mentagrophytes. For R. oryzae and S. schenckii, with the exception of R. oryzae growing in microcultures, incorporation of "4C also defined lag, exponential, and stationary growth after selection of the appropriate isotope-specific activity. This incorporation correlated directly with absorbance. We conclude that in situ microspectrophotometry, fragmented mycelium absorbance density, and, to a lesser extent, 14C incorporation can be used to effectively monitor filamentous fungal growth.