Development and Use of a Polyvalent Conjugate to Differentiate Histoplasma capsulatum and Histoplasma duboisii from Other Pathogens

Previous investigations have demonstrated the existence of five Histoplasma capsulatum serotypes. Available specific fluorescent-antibody reagents stain only four of the five serotypes. Antibodies produced against the most complete H. capsulatum serotype were labeled with fluorescein isothiocyanate to develop a reagent specific for H. capsulatum that was reactive with all the known serotypes. The unadsorbed reagent not only stained all the H. capsulatum serotypes, but it also stained cultures of Blastomyces dermatitidis, H. duboisii, several Candida species, and a variety of other fungi. Adsorption of the conjugate with antigens of C. albicans produced a reagent that intensely stained only H. capsulatum, H. duboisii, and B. dermatitidis. Differentiation of B. dermatitidis from the Histoplasma species was accomplished by application of a B. dermatitidis specific fluorescent antibody to antigens positive with the H. capsulatum reagent. At present, differentiation of H. capsulatum from H. duboisii may be accomplished only by animal inoculation. Our data substantiate the antigenic relationships hypothesized earlier, and they indicate that H. capsulatum shares at least two antigens with the other fungi that were studied.     

Monosaccharide and Chitin Content of Cell Walls of Histoplasma capsulatum and Blastomyces dermatitidis

Cell walls of Histoplasma capsulatum and Blastomyces dermatitidis, obtained by mechanical breakage of yeast- and mycelial-phase cultures, were lipid-extracted and then fractionated with ethylenediamine. Unextracted cell walls, lipid-extracted cell walls, and the three fractions resulting from ethylenediamine treatment were examined for monosaccharide and chitin content. The yeast-phase cell walls of five strains of H. capsulatum fell into two categories, designated chemotypes I and II, one of which, chemotype II, was similar to yeast-phase cell walls derived from three strains of B. dermatitidis. H. capsulatum chemotype I cell walls were characterized by lower content of material soluble in ethylenediamine, higher chitin content, and lower monosaccharide content than H. capsulatum chemotype II or B. dermatitidis cell walls. Approximately 80% of the monosaccharides of chemotype I cell walls was combined in forms susceptible to attack by mild acid hydrolysis, compared with about 50% of the monosaccharides of chemotype II and B. dermatitidis. H. capsulatum and B. dermatitidis yeast-phase cell walls could be distinguished, however, by their susceptibility to attack by a crude enzyme system derived from a Streptomyces sp. incubated with chitin as the only carbon source. Both glucose and acetylglucosamine were released from H. capsulatum cell walls, regardless of chemotype, during enzymatic hydrolysis, whereas only acetylglucosamine was released from B. dermatitidis yeast-phase cell walls. Mycelial-phase cell walls of H. capsulatum and B. dermatitidis were characterized by lower content of material soluble in ethylenediamine, higher proportions of mannose, and lower chitin content than their respective yeast phases. Glucose and acetylglucosamine were both released from all mycelial-phase cell walls, whether H. capsulatum or B. dermatitidis, by the crude enzyme system.     

In vitro interactions between Blastomyces dermatitidis and other zoopathogenic fungi

The results of in vitro interactions between colonies of Blastomyces dermatitidis and six other zoopathogenic fungi are reported. The interactions were found to range from neutral with Histoplasma capsulatum and Candida albicans to strongly antagonistic with Microsporum gypseum, Pseudallescheria boydii, and Sporothrix schenckii, and including lysis by Cryptococcus neoformans. These observations suggest that interactions between zoopathogenic fungi may be one of the biotic factors likely to influence the occurrence of B. dermatitidis in natural systems.     

Evaluation of Systemic Antifungal Agents in X-Irradiated Mice

The effect of X irradiation on the survival time of animals experimentally infected with pathogenic fungi was studied, and the activity of antifungal agents in pre-irradiated hosts was evaluated. A 24-hr preinfection dose of X irradiation decreased the survival time of mice infected with Cryptococcus neoformans and Histoplasma capsulatum to a greater extent than Candida albicans or Blastomyces dermatitidis infections. Exposure to 400 r caused a significant reduction in the variation (S(2)) survival time of C. albicans or H. capsulatum mouse infections. A single 100-mg/kg dose of 5-fluorocytosine or amphotericin B administered within 24 hr postinfection significantly extended the survival time of mice infected with C. albicans. Delayed treatment with amphotericin B was effective against C. neoformans infections. Four 50-mg/kg doses of 5-fluorocytosine were more effective than a single 200-mg/kg dose against C. neoformans infections. A single dose of amphotericin B provided significant protection when administered 48 hr postinfection against B. dermatitidis in preirradiated mice. A single dose of saramycetin 48 hr postinfection was highly effective against H. capsulatum mouse infections. A 100-mg/kg dose of amphotericin B was only effective against this fungal pathogen when administered within 8 hr postinfection. In vivo activity of the antifungal agents studied was detected within 8 to 14 days. The relative in vivo activity of several antifungal agents indicated the importance of considering their individual pharmacological properties for optimum effectiveness. The experimental model used in this study should be useful for the detection and for the preclinical evaluation of new antifungal agents.     

Amphotericin Pharmacophobia

Five cases are described in which fear of the possibly hazardous effects of giving amphotericin to patients with kidney disease resulted in death from progressive infection by an amphotericin-sensitive fungus (Cryptococcus neoformans in three cases, Blastomyces dermatitidis in one case, and Histoplasma capsulatum in one case).     

Viability, morphological characteristics and dimorphic ability of fungi preserved by different methods

The viability, morphological characteristics and dimorphic ability of fungi were evaluated. Strain subcultures were maintained under mineral oil and in soil for different periods of time, ranging from 49 to eight years. Of the 16 Blastomyces dermatitidis strains, four maintained viability and were able to complete the dimorphic process to the M phase producing a large amount of conidia, but were unable to form Y cells at 36 degrees C. Of the 15 Histoplasma capsulatum var. capsulatum strains, only one was viable but it was impossible to check its identity because it lost sporulating and dimorphic ability. Of the 53 Sporothrix schenckii strains, 37 were viable, 28 able to sporulate and 12 of them completed the whole M <=> Y dimorphic process. All subcultures in soil became inviable. The results demonstrate that the preservation methods used here affected the morphology and sporulating and dimorphic ability of the strains. B. dermatitidis and S. schenckii were considered to be species that survive better than H. capsulatum var. capsulatum under mineral oil. Thus, it is necessary to establish routine monitoring and appropriate environmental and culture conditions, using less widely spaced transplants and choosing the exact time of intervention to induce growth and development restriction in each strain.     

Mycelial- to yeast-phase transitions of the dimorphic fungi Blastomyces dermatitidis and Paracoccidioides brasiliensis.

The physiological changes that occur during the mycelial- to yeast-phase transitions induced by a temperature shift from 25 to 37 degrees C of cultures of Blastomyces dermatitidis and Paracoccidioides brasiliensis can be divided into three stages. The triggering event is a heat-related insult induced by the temperature shift which results in partial uncoupling of oxidative phosphorylation and declines in cellular ATP levels, respiration rates, and concentrations of electron transport components (stage 1). The cells then enter a stage in which spontaneous respiration ceases (stage 2), and finally, there is a shift into a recovery phase during which transformation to yeast morphology occurs (stage 3). Cysteine is required during stage 2 for the operation of shunt pathways which permit electron transport to bypass blocked portions of the cytochrome system. The mycelial- to yeast-phase transitions of these two fungi are very similar to that of Histoplasma capsulatum. Therefore, these three dimorphic fungal pathogens have evolved parallel mechanisms to adjust to the temperature shifts which induce these mycelial- to yeast-phase transitions.     

Significado del dimorfismo de ciertos hongos parasitos

Author thinks that dimorphism of certain pathogenic fungi (e.g.,Coccidioides immitis, Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporotrichum schenckii), as it is currently known, is a biological nonsense. He postulates the existance, in the extra-human life of these species, of unknown circumstances under which dimorphism would be a useful and (perhaps) necessary mechanism for surviving and thriving.     

Growth of dimorphic human pathogenicfungi on media containing cycloheximide and chloramphenicol

Summary Studies of 24 strains ofBlastomyces dermatitidis confirmed previously published results that the yeast-phase of this fungus is more sensitive than the mycelial-phase to cycloheximide and chloramphenicol.Studies of 5 strains each ofHistoplasma capsulatum, Paracoccidioides brasiliensis andSporotrichum schenckii show that that these species also have a similar yeast-phase mycelial -phase sensitivity differential in regard to these antibiotics.A cycloheximide resistant strain ofB. dermatitidis was developed from a sensitive strain.The experimental results support the general practice of using 0.5 mg/ml cycloheximide and 0.05 mg/ml chloramphenicol in media for the isolation of the four fungi at 25° C. The results indicate, however, that some strains would not be recovered at 37° C with similar concentrations of these antibiotics.It is recommended that a concentration of not more than 0.2 mg/ml chloramphenicol should be used to preserve sputum which is subsequently to be cultured forB. dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis orS. schenckii.     

In-vitro studies with SF 86-327, a new orally active allylamine derivative

SF 86-327 (Sandoz Forschungsinstitut) is an orally active allylamine derivative related to naftifine. The antifungal activity of SF 86-327 was compared in vitro with those of naftifine, ketoconazole, and itraconazole (R 51,211, Janssen Pharmaceutica) by agar dilution. 120 fungal isolates were tested. Also, the antifungal activities of SF 86-327 and naftifine against 18 dimorphic pathogens were assayed in vitro by broth dilution. Results of these studies support claims that SF 86-327 is a broad spectrum antifungal agent. Results of the broth dilution studies also revealed that SF 86-327 was both fungistatic and fungicidal in vitro for isolates of Blastomyces dermatitidis, Histoplasma capsulatum and Sporothrix schenckii at concentrations as low as 0.05 micrograms ml-1 (18 isolates tested, MIC90 = 0.39 micrograms ml-1, MFC90 = 12.5 micrograms ml-1).     

Ultrastructural Changes During the Yeastlike to Mycelial-Phase Conversion of Blastomyces dermatitidis and Histoplasma capsulatum

Fine details of the sequential anatomical events occurring during yeast to mold morphogenesis of the dimorphic fungal pathogens Blastomyces dermatitidis and Histoplasma capsulatum as seen in ultrathin sections are described and illustrated by electron micrographs. Discrete intracytoplasmic membrane systems intimately associated with the plasma membrane were observed to be formed within 6 to 8 hr after induction of the conversion process. Within 12 to 18 hr, an intermediate or transitional cell with Woronin bodies at the septum was formed from the converting yeastlike cell. Both cells were noted to contain increased numbers of mitochondria. At approximately 48 hr from the initial induction of the conversion stimuli, the newly forming hyphal cells were observed to produce postconversional intracytoplasmic membrane systems seen normally in the ultrastructural organization of the fully established mycelial-phase cell. These membrane systems appear to be associated with normal septal formation. Although minor variations of time were observed in the occurrence of the sequential events, it is suggested that yeastlike to mycelial-phase conversion of these two fungal pathogens proceeds via a similar mechanism of ultrastructural reorganization.     

An overview of macrophage-fungal interactions

A review of the literature (148 references) on the interactions of fungi with polymorphonuclear cells, monocytes and macrophages is presented. The interactions of Aspergillus species, Coccidioides immitis, Blastomyces dermatitidis, Histoplasma capsulatum, Cryptococcus neoformans, Candida albicans, and Candida species with human and experimental animal derived immune cells are examined in this overview. An effort has been made to present the reader with a comprehensive list of references with the intent of encouraging additional reading and research in this important area.     

Comparative study of a new Chrysosporium species with Histoplasma capsulatum

The Chrysosporium state of a new ascomycete, Renispora flavissima (Gymnoascaceae), resembles Histoplasma capsulatum in its macroconidial morphology. It was discovered growing in bat guano, from which it was readily isolated by direct plating of diluted soil, but only rarely from mice inoculated with soil suspensions. The fungus was consistently reisolated from tissues of mice inoculated intravenously and intraperitoneally with conidial and mycelial suspensions from cultures of the fungus. Nevertheless, there is no evidence that this species is pathogenic. Cultures grew at 37 degrees C, but did not convert to a yeast form on agar media or within cultured mouse peritoneal macrophages. Although the hyphae and conidia of this fungus fluoresce when stained with H. capsulatum fluorescent antibodies, exoantigens of the fungus produce neither H nor M precipitin bands, thus differentiating it from H. capsulatum. Both H. capsulatum and the new Chrysosporium sp. demonstrate isozyme polymorphism, and isozymic differences have been discussed between the two species.     

Serological Response of Rhesus Monkeys to Histoplasma, Blastomyces, and Coccidioides Antigens

Fifteen adult rhesus monkeys were inoculated with nonviable Histoplasma capsulatum, Blastomyces dermatitidis, or Coccidioides immitis. Antibody assays were made periodically during a 2-year period by use of a complement-fixation (CF) test employing four antigens and a latex-agglutination test. Selected sera were also studied in an immunodiffusion test and a coccidioidin-precipitin test. The serological patterns obtained with the anti-Histoplasma, anti-Blastomyces, and anti-Coccidioides monkey sera were comparable to those of sera from patients with diseases caused by the respective organisms. Rhesus monkeys should provide a good laboratory model for additional studies, including the influence of multiple antigenic stimuli on serologic response and patterns. Monkeys could also be used for the production of antisera required for studies to improve the specificity of the currently available CF antigens.     

Antigenic relationships between Aracoccidioides loboi and other pathogenic fungi determined by immunofluorescence

Summary The fluorescent antibody technique was used to study antigenic relationships betweenParacoccidioides loboi and other pathogenic fungi. The findings suggest thatP. loboi is more closely related antigenically to certainP. brasiliensis strains than to others and that it has antigens in common with the yeast form ofHistoplasma capsulatum, H. duboisii, Blastomyces dermatitidis, Candida albicans and also the mycelial form ofCoccidioides immitis. Serum globulins from 3 cases of keloidal blastomycosis were labelled with fluorescein isothiocyanate. These conjugates showed slight or no reactivity withP. loboi, the yeast forms ofP. brasiliensis, H. capsulatum, H. duboisii andB. dermatitidis, However, they stained brightlyC. albicans, serotypes A and B, the tissue form ofC. immitis and the yeast form ofSporotrichum schenckii. Adsorption of these reagents withC. albicans eliminated all staining except that forS. schenckii. These patients had no history of clinical sporotrichosis.Deceased. Last address: Fundacão Gonçalo Moniz, Salvador, Bahia, Brazil. Requests for reprints should be sent to Dr.William Kaplan.Dr.Miranda is in private practice in Rio de Janeiro, Brazil.     

Genetic diversity of Histoplasma capsulatum strains in Brazil

This study establishes the genetic relatedness among Brazilian Histoplasma capsulatum samples obtained from different sources. A PCR-based random amplified polymorphic DNA (RAPD) assay was used to delineate polymorphisms among isolates in geographically diverse regions in Brazil. RAPD fingerprints revealed distinct DNA profiles and provided a high level of discrimination among H. capsulatum strains from different locations. Cluster I was composed of H. capsulatum isolates from the northeast region. The majority of strains from southeast and south were categorized as major cluster II. The strain 84564 from Rio de Janeiro State showed no genetic correlation to any of the isolates from the same state. The RAPD patterns of H. capsulatum isolates from Goias (Cluster III) were unrelated to DNA fingerprints observed among the other H. capsulatum strains (48% similarity). This study is the first report that stratifies the clusters of H. capsulatum strains from Brazil by molecular typing and associates them with the geographical origin.     

A murine monoclonal antibody exhibiting high species specificity for Histoplasma capsulatum var. capsulatum.

A monoclonal antibody (mAb) exhibiting a high degree of species specificity for the yeast phase of the dimorphic fungus Histoplasma capsulatum was produced by a modification of the standard mAb production protocol. The technique for generating mAbs involved the use of the immunosuppressive drug cyclophosphamide to diminish the response in mice to immunodominant cross-reactive epitopes. This mAb exhibited clear specificity and did not react by ELISA with the closely related genera Blastomyces, Paracoccidioides and Sporothrix. In Western blots it recognized a linear determinant on a 70-75 kDa molecule in H. capsulatum antigen, with an extremely faint reactivity to antigens of identical molecular mass derived from Sporothrix and Paracoccidioides, and no reactivity against Blastomyces antigen.