Dennis M. Dixon

Pathogenicity and virulence of wild-type and melanin-deficient Wangiella dermatitidis

Wild-type, dematiaceous Wangiella dermatitidis (DMD 368) and melanin-deficient mutant (Mel 3) strains derived therefrom were compared for pathogenic and virulent effects in Swiss albino mice following intravenous infection. Parameters examined were mouse survival and central nervous system signs of infection, time-course cultures of fungus from brains, lungs, livers, spleens and kidneys, and histopathology of brains. Over a range of concentrations, DMD 368 produced 100% mortality while one Mel 3 strain, DMD 369, produced no mortality by 21 days after inoculation. However, in chronic infections with DMD 369, mice developed ataxia and torticollis. These signs of disease were indistinguishable from those produced by low concentrations of DMD 368. The brain was the most severely affected organ where both DMD 368 and 369 grew exponentially. Histological responses to the two strains appeared to be indistinguishable. However, the mutant appeared not to form the invasive hyphal forms of growth associated with the acute, fatal infections caused by the wild type. Thus, although the absence of melanin was associated with decreased mortality in mice, the chronic neurological signs of mouse phaeohyphomycosis appeared to be unrelated to melanin.

Dematiaceous fungal pathogens isolated from nature

This study was conducted to demonstrate the presence of pathogenic dematiaceous fungi in nature. Using hamster and mouse inoculation techniques, 43 isolates of dematiaceous fungi were recovered from 39 samples of woody plant material and soil from the Virginia environment. Thirteen species were identified and included 4 Phialophora spp., 3 Cladosporium spp., 2 Exophiala spp., Sporothrix sp., Wangiella dermatitidis, Bispora betulina, and Scytalidium lignicola. Evidence is presented for the first isolations of C. trichoides from nature in the United States; these isolates proved to be pathogenic for mice in which they produced disease and death in a course similar to that seen in man. Natural isolates of Phialophora verrucosa, Phialophora repens, Exophiala jeanselmei, and Wangiella dermatitidis were identical to those species isolated from man using the following criteria: morphology, 12% gelatin reaction, and survival in laboratory animals.

In vitro and in vivo Drug Studies with Three Agents of Central Nervous System Phaeohyphomycosis

Amphotericin B (Amph B), 5-fluorocytosine (5-FC), ketoconazole (KTZ), fluconazole (FLZ), amorolfine (AMOR) and terbinafine (TER) were tested against 3 agents of central nervous system phaeohyphomycosis in vitro and in life-threatening infections in mice. The fungi studied were Cladosporium bantianum, Dactylaria constricta and Wangiella dermatitidis. The broadest protection against this group of fungi in mice was offered by 5-FC followed by Amph B and FLZ, then KTZ. AMOR and TER were inactive in vivo. The results of in vitro susceptibility testing had no predictive value. In contrast, the data obtained from the mouse models should be useful clinically.

Human Infectious Corneal Ulcer Caused by Pythium Insidiosum

Pythium insidiosum is a fungus-like organism known to infect a variety of animals. In humans, the few known cases involving Pythium have included arterial infections and cellulitis. We present what we believe to be the first case of P. insidiosum recovered from a human corneal ulcer. The organism is difficult to isolate, causing delays in diagnosis. It is also resistant to the usual antifungal medications, making surgical excision the treatment of choice.

Experimental central nervous system phaeohyphomycosis following intranasal inoculation of Xylohypha bantiana in cortisone-treated mice

Experimental central nervous system (CNS) phaeohyphomycosis was established in cortisone-treated mice following intranasal exposure to conidia of Xylohypha bantiana (Cladosporium bantianum, C. trichoides). X. bantiana was recovered from the lungs of 78% of intranasally inoculated normal mice sacrificed within the first 3 days of infection and from 15% at day 28. The fungus was not recovered from the brains of normal mice. In contrast, X. bantiana was recovered from only 33% of the lungs of cortisone-treated mice within the first 3 days of infection. However, the fungus was recovered from the brains of 11% of cortisone-treated mice sacrificed or dying over a 28 day period. Histologically and temporally the CNS disease in cortisone-treated, intranasally inoculated mice was consistent with hematogenous dissemination from a primary pulmonary focus.

Mel- mutants of Wangiella dermatitidis in mice: evaluation of multiple mouse and fungal strains

Melanin-deficient mutants of Wangiella dermatitidis were studied by comparing a spontaneous mutant (Mel 3) with the parental wild type (wt) in four strains of mice and by comparing various UV-induced mutants in a single strain of mouse. Mice were inoculated intravenously with 3 x 10(6), 1 x 10(7), 3 x 10(7) or 1 x 10(8) yeast-like cells and mortality assessed at the end of 20 days. Neuropathology was evaluated in methenamine-silver stained brain sections of the different strains of mice injected with either wt or Mel 3 (1 x 10(7) cells per mouse). In general, both fungus strain related and mouse strain related differences in mortality were observed. The DBA/2J mouse was the most susceptible to fatal infection with all fungus strains. In the other strains of mice, however, the melanin deficient mutants were significantly less virulent than the wt at a concentration of less than or equal to 3 x 10(7) cells per mouse. No obvious trend was seen in the numbers of brain lesions in different strains of mice with respect to wt vs. Mel 3. However, invasive hyphal forms did seem to be associated with virulence.

Comparative histopathology of Dactylaria constricta, Fonsecaea pedrosoi, Wangiella dermatitidis, and Xylohypha bantiana in experimental phaeohyphomycosis of the central nervous system

Summary: The comparative neuropathology of experimental phaeohyphomycosis due to Dactylaria constricta (D. gallopava; Scolecobasidium constrictum), Fonsecaea pedrosoi, Wangiella dermatitidis (Exophiala dermatitidis), and Xylohypha bantiana cladosporium bantianum; C. trichoides), was studied in the mouse central nervous system (CNS). All fungi appeared as brown hyphae on hematoxylin and eosin stain. Zigzag forms of hyphae were common in all species except F. pedrosoi. Neither blastoconidia nor chlamydoconidia were present in tissue infected with D. constricta. D. constricta, W. dermatitidis, and X. bantiana were distributed hematogenously throughout the cerebral hemispheres and demonstrated intraluminal, microvascular invasion whereas, CNS infection due to F. pedrosoi developed by extension from extracerebral dural lesions. F. pedrosoi invaded small to medium‐sized cerebral arteries and veins by extraluminal extension. Microabscesses and early granuloma formation occurred with D. constricta, W. dermatitidis, and X. bantiana, but not with F. pedrosoi, which was associated with only scant cellular inflammatory reaction. Only D. constricta caused a necrotizing ventriculitis with periventricular hemorrhage. Other than yeast‐like cells in W. dermatitidis and F. pedrosoi, ventriculitis in D. constricta, and epidural invasion in F. pedrosoi, these infections are remarkably similar in tissue.

In vivo and in vitro studies with an atypical, rhinotropic isolate of Cryptococcus neoformans.

An atypical isolate of Cryptococcus neoformans was investigated because of its consistent and reproducible production of gross nasal pathology following i.v. injection in Swiss albino mice. Dose response to graded concentrations ranging from 1×l02–1×l07 cells/mouse yielded an LD50 of 1.4×103 cells/mouse for the atypical rhinotropic strain H140 which was significantly less virulent (p<0.01) than our reference strain of Cryptococcus neoformans. There was no significant difference in mortality following the injection of in vitro vs. in vivo passed inoculum. As early as two weeks after inoculation, this strain produced gross nasal enlargement to approximately 2–3 × normal dimensions with granulomatous and ulcerated lesions. The LD60 resulted in the greatest percentage of nasal involvement (85%). C. neoformans was demonstrated by culture and histopathology in the noses, brains, lungs, livers and kidneys. A temperature selection was indicated by findings of a lower temperature minimum for subcultures isolated from the noses relative to those isolated from the brain, and by the fact that the most densely populated organs following intraperitoneal injection were the testes. This route of inoculation resulted in cutaneous nasal involvement in a manner analogous to that following i.v. injection. The atypical isolate was unable to assimilate trehalose or raffinose but otherwise was entirely consistent with identification as C. neoformans and produced characteristic CNS and general organ system disease in addition to the rhinotropic cutaneous manifestations. The model characterized here in normal mice may be of value in studies of fungal dermotropism.

A comparison of bifonazole (bay h 4502) with clotrimazole in vitro

The antifungal activity of a new topical imidazole, bifonazole (BAY h 4502, Bayer AG Institute for Chemotherapy), was compared in vitro with that of clotrimazole (BAY b 5097, Schering Corporation) in tests with 67 pathogenic and commensal yeasts, 45 dermatophytes and 14 miscellaneous pathogenic fungi by an agar dilution method. Three media, Kimmigs agar, Sabourauds agar, and casein-yeast extract-glucose agar were used. Bifonazole was inhibitory for nearly all the yeasts tested including Candida albicans, C. parapsilosis, and Torulopsis glabrata with geometric mean minimal inhibitory concentrations (G-MIC) averaging 5 micrograms ml-1 on all three media. Clotrimazole was the more active drug against these same species with G-MICs ranging from 0 . 25 to 2 . 10 micrograms ml-1. Results with bifonazole were affected by choice of medium with Kimmigs agar generally giving the lowest MICs; results with clotrimazole were also affected by choice of medium but to a lesser degree. In nearly all instances, both bifonazole and clotrimazole were inhibitory for the dermatophytic fungi at concentrations of 0 . 50 micrograms ml-1 or less and clotrimazole was the more active drug. Choice of medium was, in general, not a factor with these latter fungi which included Epidermophyton, Trichophyton, and Microsporum species. Both drugs were active against species of Aspergillus (G-MICs of 3 . 18 micrograms ml-1), Fusarium (G-MICs ranging from 1 . 59 to 12 . 70 micrograms ml-1) and Scopulariopsis (G-MICs of 1 . 78 micrograms ml-1); clotrimazole was the more active drug by factors of 2- to 4-fold on all three media. Bifonazole MICs were shown to vary with pH (maximal activity at pH 6 . 5) with selected yeasts when tested on Kimmigs agar. Differences in results obtained with varying inoculum sizes for these same yeasts generally were unremarkable. With selected species of yeasts and dermatophytes, it was determined that the ratio of minimal fungicidal to inhibitory concentrations (MFC/MIC) was much lower for bifonazole than for clotrimazole.

Dactylaria constricta: Another dematiaceous fungus with neurotropic potential in mammals

Dacylaria constricta, a neurotropic dematiaceous fungal pathogen of poultry, was evaluated for pathogenic potential in mice. Four isolates of D. constricta and two of Scolecobasidium humicola were injected intravenously into groups of mice. Two isolates of a D. constricta group formerly known as Dactylaria gallopava produced signs of central nervous system infection with cerebral microabscesses and death. None of the mice injected with the two remaining isolates of D. constricta from a group formerly known as Scolecobasidium constrictum or with isolates of S. humicola showed any evidence of infection, and none died. Potential pathogenicity can now be added to the previously established physiological differences to separate isolates of D. constricta into two distinct groups.