Diagnosing coccidioidomycosis outside an endemic area. A recent case in Montreal, Quebec, Canada.

A 65-year-old female with a long-standing controlled Crohn's disease was admitted in August 1979 to a Montreal hospital for investigation of a non-calcified RUL lung nodule. After an extensive work-up (negative bronchoscopy and mediastinoscopy showing a granulomatous reaction) she underwent thoracotomy; the nodule was enucleated and showed caseous material within a fibrous capsule. Cultures in mycology produced a heavy growth of Coccidioides immitis, a diphasic pathogenic fungus endemic in South Western USA, identified by its morphology in autoclaved cultures, reversion to the spherule phase in mice and a 1:80 immunofluorescent stain with both commercial and patient's serum. The patient, who travelled to Arizona three times in recent years, was soon discharged and remains apparently well. Because of inherent risks from handling cultures of C. immitis, the authors urge that, when coccidioidomycosis is considered clinically and epidemiologically, the pertinent information be transmitted to the laboratory.

First case of human protothecosis in Canada: laboratory aspects.

A case of bursitis due to Prototheca wickerhamii is briefly reported. In histological sections the organism stained well with fungal stains, grey with silver methanamine and red with periodic acid Schiff reagent. This unicellular achlorophyllous alga was studied on common laboratory media. The characterization of the Prototheca sp. depends largely on wet mount microscopic examination from broth or agar cultures which ensures the observation of endosporulation and a consistent absence of budding. Otherwise the growth rate and the pasty white colonies may lead to an erroneous identification, most likely as a Cryptococcus sp. P. wickerhamii lends itself very well to standard physiological tests used for the identification of yeasts. The strain was found insensitive to 5-fluorocytosine. The MIC of amphotericin B was 0.15 microgram/ml.

On the occurrence of Microsporum persicolor in Montreal, Canada.

A case of Microsporum persicolor skin infection in a casual employee of a veterinary clinic in Montreal, Quebec, Canada, is reported. A survey of 80 wild rodents in the Montreal area resulted in the isolation of M. persicolor from two voles (Clethrionomys gapperi) and one deer mouse (Peromyscus maniculatus). One of the voles, heavily infested with parasites including ticks, had obvious lesions on the nose. This is the first report of M. persicolor in rodents in North America.

Isolation of dermatophytes, Candida species and systemic fungi from dermatologic specimens in Montréal, 1963 to 1973.

Of 10 057 specimens of scrapings from skin, nails and scalp examined for dermatophytes, yeasts, pityriasis versicolor and systemic mycoses between 1963 and 1973, 30.4 percent were positive for fungi. Skin produced the highest proportion (68.6 percent) of positive scrapings, scalp the lowest (4.2 percent). Trichophyton rubrum was the predominant species (23.6 percent); of lesser prevalence were Microsporum canis (9.3 percent), T. mentagrophytes (8.4 percent) and Epidermophyton floccosum (4.8 percent). Double infections were encountered on 102 occasions; T. rubrum and Candida parapsilosis were the most frequent cohabiting species. The introduction in 1966 of periodic acid-Schiff staining for routine examination of scrapings resulted in better diagnostic results, particularly in the case of culturally nonproductive specimens and cases of pityriasis versicolor. Blastomyces dermatitidis and Cryptococcus neoformans were isolated from two patients in the course of routine investigation for dermatophytes.

Yeast-like growth of Mucor alternans (van Tieghem) in tissue-culture medium 199.

Mucor alternans(van Tieghem) was found to be a diphasic species in that it grew exclusively in the yeast-like budding phase under anaerobic conditions in the complex medium yeast extract-peptone-glucose broth and in tissue-culture medium 199. In the latter medium this growth form occurred also at 37C, at an initial pH of 7.2, and at glucose concentrations of 0.1 and 1.0%. The authors suggest that because of its synthetic nature the tissue-culture medium could be used with advantage in the study of nutritional requirements of dimorphic mucors.

Production of diagnostic pigment by phenoloxidase activity of cryptococcus neoformans.

Cryptococcus neoformans produces brown pigmented colonies when grown on agar media made from an extract of potatoes and carrots, broad beans (Vicia faba), or Guizotia abyssinica seeds. Since other yeasts do not produce the pigment, these media are useful as differential isolation media for C. neoformans. Similar specific pigment was produced by C. neoformans on chemically defined agar media which contained six different substrates of phenoloxidase (o-diphenol: oxygen oxidoreductase EC 1.10.3.1) an enzyme which catalyses the oxidation of o-diphenols to melanin. Substrates were incorporated singly into the media and included L-3, 4-dihydroxyphenylalanine (L-DOPA), chlorogenic acid, protocatechuic acid, catechol, norepinephrine, and 3-hydroxytyramine hydrochloride (dopamine). No pigment was produced on media without substrate. Phenoloxidase activity in (NH(4))(2)SO(4) precipitates of C. neoformans cell-free extract was assayed by measuring increases in absorbance at 480 nm produced in solutions of L-DOPA. This reaction showed oxygen uptake and was effectively inhibited by copper chelators, but not by catalase. The enzyme also oxidized the five other substrates which induced pigment formation. Electron micrographs of cells incubated in L-DOPA showed deposition of the pigment in the cell wall.

Improvement in laboratory diagnosis of pulmonary cryptococcosis.

A new differential colour medium prepared with an extract of potatoes and carrots produced ochre-brown pigmentation in Cryptococcus neoformans colonies, identical to that obtained with Guizotia abyssinica seed extract. The pigment formation was observed to be specific for this pathogenic yeast in a survey of 534 yeast-positive specimens during routine diagnostic work; among 47 C. neoformans-positive specimens were 27 sputa and bronchial aspirations, many overgrown by common yeasts.Detection of C. neoformans in the contaminated specimens was credited largely to the differential medium used. Twelve cases of cryptococcosis were investigated between 1962 and 1968, five of which presented involvement of the respiratory tract only.

Inhibition of Histoplasma capsulatum by Candida albicans and other yeasts on Sabouraud's agar media.

The inhibition of growth of Histoplasma capsulatum by Candida albicans and other yeasts on Sabouraud's agar was investigated. Histoplasma (yeast-phase inoculum) was grown alone and in mixtures with yeasts at 25 C for 4-week periods. As few as 10 colonies of C. albicans completely inhibited the growth of approximately 50,000 potential colonies of Histoplasma. The pH was determined in cultures of 36 colonies of Candida on media containing 1, 2, and 4% glucose by spotting the agar with pH indicators. A drop in the pH became noticeable in all three media about the 3rd day of incubation, and a pH of 3.5 was reached in about 7 days. Subsequently, the pH remained almost stationary in the 4% glucose-agar, rose slowly in the 2% glucose-agar, and rose sharply in the 1% glucose-agar. The growth of Histoplasma was inhibited completely at pH 4 and below. When the pH was controlled in mixed cultures, some growth of Histoplasma was obtained. Substitution of maltose for glucose delayed the development of acidity and allowed the appearance of numerous mycelial colonies in the presence of Candida. This growth was arrested as soon as the medium became acid. Four other species which also acidified the Sabouraud's medium effected similar inhibition. It was thus shown that severe and prolonged acidity produced by some yeasts in the sugar-rich Sabouraud's media is alone sufficient to completely inhibit Histoplasma during the standard 4-week incubation of specimens such as sputum.