Monitoring of filamentous fungal growth by in situ microspectrophotometry, fragmented mycelium absorbance density, and 14C incorporation: alternatives to mycelial dry weight.

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-14C]glucose. Microcultures (200 microliter) 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 14C 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.

Chronic dermatophyte infection: evaluation of the Ig class-specific antibody response reactive with polysaccharide and peptide antigens derived from Trichophyton mentagrophytes.

An enzyme-linked immunosorbent assay was used to detect serum antibodies within Ig classes IgG, IgM, IgA, and IgE against two partially purified Trichophyton mentagrophytes-derived antigens: polysaccharide (SAC) and peptide (PEP). Sera from 27 chronically infected adults, 16 normal noninfected adults, and 17 noninfected children were evaluated. All sera were reactive, indicating that circulating antibody reactive to PEP and SAC is a feature common to most people, regardless of age and the presence of active infection. The reactivity to SAC was much greater than to PEP. Inhibition assays revealed that components of PEP are found in SAC. Chronically infected adults showed slightly elevated IgG and IgA reactivity to both SAC and PEP compared with noninfected normal adults. This elevated reactivity correlated with the extent (% surface affected) of cutaneous infection. IgE antibody reactivity was detected only against SAC and was somewhat elevated in chronically infected adults. There was no association between antibody reactivity and immunoglobulin level or between the individual subject's level of antibody reactivity to PEP and SAC.

Orally administered ketoconazole: route of delivery to the human stratum corneum.

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.

Fungistatic mechanism of human transferrin for Rhizopus oryzae and Trichophyton mentagrophytes: alternative to simple iron deprivation.

Human serum, human transferrin (TF), and the iron chelator 1,10-phenanthroline (OP) produce iron-reversible fungistatic activity which has been attributed to simple iron deprivation. In this study, the influence of the size of the inoculum on the inhibitory activity of serum, TF, and OP prepared with the same iron-binding capacity (2.5 micrograms/ml) for Rhizopus oryzae and Trichophyton mentagrophytes was examined. Inhibition was monitored in liquid microcultures maintained at 37 degrees C and pH 7.4 to 7.5 by measuring the change in absorbance density. Increasing the number of spores in the inoculum disrupted the fungistatic activity of serum and TF, but not that of OP. The dilution at which OP lost fungistatic activity was not affected by the number of spores in the inoculum and was the same for both fungi. The dilution at which TF and serum lost fungistatic activity was dependent upon both the quantity of the inoculum and the species of fungus. The number of viable spores, rather than the total number of spores in the inoculum, was determined to be important in overcoming the inhibition of fungal growth by serum and TF. The fungistatic activity of serum and TF could be diminished by the preexposure of the serum to viable but nongrowing spores. Direct and indirect fluorescence studies indicated that both T. mentagrophytes and R. oryzae absorbed TF. Glucose uptake by R. oryzae was inhibited by a 4-h exposure to 5.0 to 0.15 mg of apotransferrin per ml. These results suggest that the fungistatic activity of TF for R. oryzae and T. mentagrophytes may not be attributable to simple iron deprivation and raise the possibility of a requirement for a direct interaction.

Lyophilized microculture susceptibility test for ketoconazole, miconazole, clotrimazole, and griseofulvin against dermatophytes.

A lyophilized microculture antimycotic susceptibility testing system for ketoconazole, miconazole, griseofulvin, and clotrimazole is described. Microculture plates were loaded with 100 microliters of medium and 10 microliters of appropriate concentrations of the four antimycotics and were lyophilized to complete dryness. The lyophilized plates were stored at -70 degrees C or 4 degrees C or in a desiccator at 25 degrees C. Samples from each storage condition were rehydrated at 1, 2, 3, 4, 5, 6, 8, 10, and 12 months and inoculated with Trichophyton mentagrophytes (Robin) Blanchard ATCC 18748. All of the minimal inhibitory concentrations (MICs) generated from the lyophilized microcultures were within one experimental dilution of MICs derived from fresh microcultures. The ability of reconstituted lyophilized microcultures to consistently produce MICs comparable to MICs derived from fresh microcultures was characterized. Nine dermatophyte isolates were tested five times each over a 70-day period. The MICs derived were reproducible and comparable to MICs determined by freshly prepared microculture tests. Lyophilization of freshly prepared antimycotic-containing microcultures does not alter the MIC resolution of the testing system and provides an effective method of storage of prepared antimycotic tests for ketoconazole, miconazole, clotrimazole, and griseofulvin.

Hydroxamate siderophore production by opportunistic and systemic fungal pathogens.

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 degrees C in a chemically defined low-iron media (0.2 microM Fe). Culture supernatants were assayed for siderophores by two nonspecific methods [FeCl3 and Fe(ClO4)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 degrees C than at 37 degrees C. The present study adds eight fungi to the list of known siderophore producers and confirms siderophore production by H. capsulatum.

Liposomes containing 3-n-pentadecylcatechol induce tolerance to toxicodendron.

Pentadecylcatechol (PDC) (1 mg) incorporated into liposomes (PDC-liposomes) and given by intracardiac injection to guinea pigs 1 week prior to attempted topical sensitization to PDC significantly inhibited that sensitization as evidenced by patch tests done 2 weeks after the attempted topical sensitization. PDC (1 mg) dissolved in ethanol did not significantly inhibit sensitization. Sensitization inhibition was specific since dinitrochlorobenzene sensitization was not inhibited by prior intracardiac treatment with PDC-liposomes. In addition, the sensitization to PDC was no longer inhibited if the time between the intracardiac PDC-liposome injection and the topical PDC sensitizing dose was increased from 1 week to 2 or more weeks.

Restoration of Trichophyton mentagrophytes growth in medium depleted of metals by chelation: importance of iron.

The present study examined the requirement of Trichophyton mentagrophytes ATCC-18748 for iron. Nutrient broth depleted of iron by the chelating cation exchange resin Chelex-100 did not support the growth of T. mentagrophytes beyond germ tube formation. The soluble chelate of iron, ferric ammonium citrate, restored the capacity of the chelated medium to support fungal growth in proportion to the amount of iron added. Ferric chloride, which rapidly becomes insoluble at neutral pH, was not effective in the medium. The soluble salts of cobalt, chromium, copper, manganese, nickel, and zinc individually did not replace the requirement for iron. A method for defining the iron requirement based upon utilization of iron from ferric ammonium citrate is described. These data indicate that the growth of T. mentagrophytes ATCC-18748 is iron-dependent, which is consistent with the hypothesis that serum transferrin inhibits dermatophyte growth by the mechanism of iron deprivation.

Factors affecting griseofulvin susceptibility testing of Trichophyton rubrum in microcultures.

A microculture broth assay system for griseofulvin susceptibility testing of Trichophyton rubrum was further characterized. The effects of mass and number of colony-forming units of a fragmented mycelial inoculum, 5- or 8-day incubation periods, 25 or 32 degrees C incubation temperatures, and the solvents used to dissolve griseofulvin on the minimal inhibitory concentration (MIC) of griseofulvin were determined. An inoculum density with an absorbance of 0.600 at 450 nm ensured successful inoculation of all microcultures. Reduction of the inoculum mass to an absorbance of 0.200 lowered the number of colony-forming units in the inoculum by 60 to 80%. This decreased the efficiency of inoculation but did not alter the resulting MIC. There was no correlation between MIC and the number of colony-forming units used to initiate growth. Neither incubation temperature nor the length of incubation affected the MIC. The use of either acetone or ethanol to solubilize griseofulvin likewise had no effect on the MIC. The mean reproducibility of the MICs determined with the microculture method was 96%.

A mechanism of susceptibility to mucormycosis in diabetic ketoacidosis: transferrin and iron availability.

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.

Fungistatic capacity of sera from guinea pigs injected with various iron solutions: differences between Trichophyton mentagrophytes and Rhizopus oryzae.

The fungistatic capacity and serum ion levels (SI) of guinea pigs given subcutaneous injections of various iron solutions were examined. The administration of 2.0 ml of 0.1 M ferric ammonium sulfate, ferric sulfate, or ferric chloride subcutaneously had no significant effect on the SI 3 h after administration, whereas ferric ammonium citrate, ferric citrate, or ferrous sulfate elevated the SI to 50 to 140 times that necessary to saturate the unbound transferrin in normal sera. The sera from 11 of 15 guinea pigs with an elevated SI remained fungistatic for Trichophyton mentagrophytes, whereas 3 of 15 guinea pigs with an elevated SI remained fungistatic for Rhizopus oryzae. The sera from normal guinea pigs were consistently fungistatic for Rhizopus oryzae. The sera from normal guinea pigs were consistently fungistatic for both T. mentagrophytes and R. oryzae. These data suggest that subcutaneous administration of certain iron compounds can significantly elevate the SI without completely abolishing the fungistatic capacity of the serum.

Oral ketoconazole. An effective and safe treatment for dermatophytosis.

This study demonstrates the efficacy and safety of ketoconazole, a broad-spectrum oral antifungal agent administered to 20 patients with severe, extensive, and recalcitrant Trichophyton rubrum infection. The average patient has had continuous infection for 20 years. Sixteen patients had glabrous skin infection that encompassed an average of 40% of their skin surface. The remaining four patients had palmar-plantar infection. Ketoconazole was administered for 27 to 70 days in a daily oral dose of 200 or 400 mg. Initial clinical and mycological response occurred within five to seven days, and the glabrous and/or palmar-plantar skin changes improved at least 90% in all patients. In 13 of these patients, the infection cleared completely. The only side effects experienced--pruritus in four patients and photophobia in two patients--did not necessitate interruption of therapy. Overall, ketoconazole was found to be an effective and safe therapy for dermatophytosis. Follow-up examinations of our cases five months later showed recrudescence in 75% of them, which was not unexpected with these severe infections.

Griseofulvin-resistant dermatophytosis correlates with in vitro resistance.

The unsuccessful treatment of dermatophytosis with griseofulvin is common. The mechanism is not known but may involve infection with a griseofulvin-resistant dermatophyte. The mean minimal inhibitory concentration (MIC) value of griseofulvin for Trichophyton rubrum isolates obtained from griseofulvin unresponsive patients was substantially larger than the mean MIC value for responsive control isolates. This difference indicates that therapeutic failure does correlate with the relative in vitro resistance. An MIC of 3.0 microgram/mL or greater was determined to indicate relative griseofulvin resistance. We conclude that the MIC determination for griseofulvin can be used to determine the appropriateness of griseofulvin therapy.

Antimycotic susceptibility testing of dermatophytes in microcultures with a standardized fragmented mycelial inoculum.

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 degrees C. The mycelia were harvested before conidia appeared and were fragmented to 10 to 50-micron 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 agent-containing 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.

The effect of human lymphokine on the growth of Trichophyton mentagrophytes.

Lymphokine was tested for fungal growth inhibitory activity against the filamentous fungus Trichophyton mentagrophytes. Human peripheral blood lymphocytes from a donor exhibiting delayed type cutaneous hypersensitivity to a trichophytin skin test were cultured with trichophytin and PHA-P. Culture supernatants were assayed for lymphokine activity using the lymphotoxin sensitive mouse L-929 alpha fibroblast. Lymphocyte activation to PHA-P and trichophytin was confirmed by monitoring 3H-thymidine incorporation. Supernatants from 2-day PHA-P and 6-day trichophytin activated cultures were found to contain potent lymphokine activity. This activity was not diminished by the addition of ferric iron sufficient to saturate the contained transferrin. Supernatants from unstimulated control cultures contained no lymphokine activity. Undiluted lymphokine containing supernatants and nonlymphokine containing control supernatants were evaluated for fungal growth inhibitory activity using a sensitive radiometric growth assay. Iron supplemented supernatants retaining potent lymphokine activity did not inhibit fungal growth. Non-iron supplemented supernatants and fresh medium containing serum inhibited fungal growth. Our data suggest that lymphokine active against mammalian cells is not directly antagonistic to the growth of the filamentous fungus T. mentagrophytes but does not exclude the possibility that activated lymphocytes release a chelator such as transferrin that can inhibit fungal growth.

The effect of zinc on the sebum secretion rate.

In accordance with a randomized double-blind experimental design, capsules of zinc sulfate, 440 mg total daily dose, and lactose placebo were administered orally to 10 normal Caucasian males for 3 weeks. Sebum secretion rates and serum zinc levels were determined prior to and following treatment. There was a statistically significant change in the before and after mean sebum secretion rates of the zinc group when compared with those of the placebo group (p less than 0.028). The results of this preliminary study indicate that supplemental zinc sulfate may reduce the quantity of skin-surface sebum. Further investigation is warranted.

Filamentous fungal growth assay: correlation between [U-14C] glucose uptake and dry weight determinations.

The filamentous fungus Trichophyton mentagrophytes was grown in [U-14C] glucose supplemented nutrient broth. Growth was monitored in 200 microliter microcultures and 5 ml macrocultures by measuring the incorporation of 14C into trichloroacetic acid-insoluble macromolecules and in macrocultures by dry weight determination. Adjustment of the specific activity of the [U-14C] glucose medium supplement from 1080 muCi/mmole to 108 muCi/mmole decreased the rate of isotope uptake and prolonged availability so that growth beyond 36 h could be monitored radiometrically. The dry mycelial weight of the fungus was directly proportional (r = 0.995) to the amount of isotope incorporated. Isotope uptake in microcultures and macrocultures was kinetically identical. These data clearly indicate that the uptake of 14C by T. mentagrophytes can be used to monitor fungal growth accurately in both macrocultures and microcultures.

Atopic dermatitis, impaired cellular immunity, and molluscum contagiosum.

A substantial elevation in the level of serum IgE (7,000 to 19,000 ng/ml) was noted in a man with atopic dermatitis and chronic molluscum contagiosum. Cell-mediated immunity (CMI) was depressed in vivo (cutaneous anergy), whereas in vitro tests showed normal numbers of "T" rosette-forming lymphocytes, a normal phytohemagglutinin-P-elicited lymphocyte transformation response, and lymphocyte transformation reactivity to the antigens streptokinase-streptodornase and purified protein derivative. Accumulated evidence suggests that patients with atopic dermatitis may have, associated with an elevated serum IgE level, a functional defect(s) in CMI that is greater in vivo than in vitro. This functional defect may impair host defense and account for the chronic molluscum contagiosum infection present in this patient.

An automated radiometric microassay of fungal growth: quantitation of growth of T. mentagrophytes.

An automated radiometric microassay of the growth of Trichophyton mentagrophytes and other filamentous fungi is described. The assay is based upon the incorporation of 14C(U) glucose into the organism. Fractionation studies indicate that 73% of the label is found in trichloroacetic acid-insoluble macromolecular components of the mycelium. Incorporation of label directly correlated with growth as estimated by visual scoring of turbidity and as recorded in photomicrographs. Incorporation of 14C(U) glucose delineated a lag, exponential and stationary or plateau phase of growth. These phases could be completely inhibited by the antifungal agent tolnaftate. It was concluded that the growth of filamentous fungi can be successfully monitored by the radiometric method described. Moreover, this method is sensitive, accurate, reproducible, rapid and free of the variability inherent in many traditional estimates growth.