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A new medium effectively differentiates B and C serotypes of Cryptococcus neoformans from A and D serotypes on the basis of resistance to low concentrations of cycloheximide and assimilation of glycine as the sole carbon source.
Two tests were developed for the rapid identification of Cryptococcus neoformans based on pigment produced by the organism's phenoloxidase activity. Caffeic acid was incorporated into cornmeal agar, a medium used routinely for yeast identification. When tested on this medium, only C. neoformans isolates produced brown pigment. All other yeasts maintained their normal morphology and did not produce the reaction product. A non-medium-based test was developed for same-day identification of C. neoformans isolates. Paper strips saturated with a buffered L-beta-3,4-dihydroxyphenylalanine-ferric citrate solution were inoculated with isolates and incubated at 37 degrees C. Pigment production occurred only with C. neoformans isolates, many within 60 to 90 min. All other yeasts remained negative.
Of 90 clinical isolates of Cryptococcus neoformans studied, 3 were determined to be serotype B/C. The patients from whom these B/C isolates were obtained were identified as never having lived in or visited the areas associated with B/C serotypes. This finding suggests a broader geographic distribution of this serotype group than previously believed. The glycine-cycloheximide-phenol red medium described by Salkin and Hurd (J. Clin. Microbiol. 15:169-171, 1982) was shown to be more accurate in differentiating A/D and B/D serotype pairs of C. neoformans than the creatinine-dextrose-bromthymol blue medium described by Kwon-Chung et al. (Int. J. Syst. Bacteriol. 28:616-620, 1978).
Several indoles served as substrates for the phenoloxidase of Cryptococcus neoformans and resulted in the production of a melanin-like pigment. In general, a higher percentage of C. neoformans var. neoformans (A and D serotypes) isolates could produce pigment from indoles than could those of var. gattii (B and C serotypes). Only compounds with a hydroxyl or an amino group on the phenyl ring produced pigment; methoxy, nitro, methyl, and fluorine substituents on the phenyl ring were inactive, as was a hydroxyl group at the 2 position on the indole ring. The phenoloxidase of C. neoformans thus appears to differ from that found in Mycobacterium leprae, which cannot use a hydroxyindole, desoxyfructo-5-hydroxytryptamine, as a substrate. In addition, C. neoformans differs from M. leprae in that desoxyfructo-5-hydroxytryptamine does not inhibit the uptake of dihydroxyphenylalanine into the cell.
For the first time, Cryptococcus neoformans serotype B was isolated from a patient in New York City, not a region endemic for B/C serotypes. The isolate was morphologically unusual, with cells several times larger in infected tissue than those characteristic of the yeastlike pathogen. This anomaly may be a problem in the identification of similar isolates. Two serotype differentiation media and a slide agglutination test were used for definitive serotype identification.
A clinical isolate of Cryptococcus neoformans was grown on essential salts medium without (NH4)2SO4 and supplemented with bovine serum albumin as the sole carbon and nitrogen source. Growth on this medium resulted in secretion of proteolytic enzymes by the organism. The secretion of protease enzymes was inhibited by (NH4)2SO4 and glucose. Proteolytic activity may be a factor in virulence and pathogenicity of C. neoformans.
An examination of 45 clinical isolates of Cryptococcus neoformans revealed an unusually high incidence of C. neoformans var. gattii in South Australia (65%) and in the Northern Territory (95%). In assessing all the available data from Australian isolates of C. neoformans, there appeared to be an endemic focus for the incidence of C. neoformans var. gattii in the rural aboriginal population of the Northern Territory.
We report a case of fungemia and disseminated disease caused by a urease-negative strain of Cryptococcus neoformans in a patient with the acquired immune deficiency syndrome. Except for failure to hydrolyze urea, the microbiological characteristics of the isolate were typical of C. neoformans. Laboratory specialists should be aware of the occurrence of atypical strains of C. neoformans, particularly those recovered from patients with the acquired immune deficiency syndrome.
We used discriminant analysis of cellular fatty acid compositions determined by gas-liquid chromatography to differentiate yeastlike fungi (a total of 190 strains; including 37 Candida albicans strains, 21 Candida krusei strains, 13 Candida guilliermondii strains, 37 Candida tropicalis strains, 10 Candida pseudotropicalis strains, 24 Candida parapsilosis strains, 32 Torulopsis glabrata strains, and 16 Cryptococcus neoformans strains). Previous results with a standard strain of C. albicans indicated that reproducible fatty acid chromatograms can be obtained with cells grown in a medium of 2% Sabouraud glucose agar at 35 degrees C for between 48 and 72 h. These conditions were also maintained in cultures of the other organisms that we studied. The cellular fatty acid compositions of the organisms were determined quantitatively by gas-liquid chromatography and analyzed by discriminant analysis. The total correct identification expressed as relative peak percent was 95.8% (89.2% for C. albicans to 100% for C. krusei, C. guilliermondii, C. pseudotropicalis, T. glabrata, and C. neoformans). The total correct identification expressed as the common peak (palmitic acid) ratio was 94.7% (87.5% for C. parapsilosis to 100% for C. pseudotropicalis, T. glabrata, and C. neoformans). Both results suggest that cellular fatty acid compositions can be differentiated by this method.
Environmental isolations have established that Cryptococcus neoformans var. gattii appears to have a specific ecological association with Eucalyptus camaldulensis. So far, we have isolated C. neoformans var. gattii on 35 separate occasions, all from samples associated with E. camaldulensis. The global distribution of E. camaldulensis appears to correspond to the epidemiologic distribution of cryptococcosis caused by C. neoformans var. gattii. No other environmental source for the fungus has yet been detected, and no other eucalypt has the distribution pattern corresponding to reported cases caused by this fungus. These findings may provided an explanation for the high incidence of infections caused by C. neoformans var. gattii in Australian aborigines living in the Northern Territory and for its low worldwide incidence in acquired immunodeficiency syndrome patients.
Cryptococcus neoformans was cultured from 13 (3%) of 469 clinical specimens examined from the respiratory tract of patients with bronchopulmonary diseases. These isolations came from 5 (2%) of 207 patients; 11 isolates were from sputum and 1 each were from bronchoscopic aspirate and empyema pus. The fungus was not cultured from the oropharyngeal washings of 101 apparently healthy volunteers. Of the 5 patients, 3 had pulmonary tuberculosis, including one with pyopneumothorax and 2 with allergic bronchopulmonary aspergillosis as the underlying disease. In the tuberculosis patient with pyopneumothorax and C. neoformans in empyema pus, the fungus was presumably a tissue invader, whereas its role could not be unequivocally ascertained in the remaining 4 patients from whom it was isolated from sputum or bronchial aspirate on at least two consecutive occasions. The question of C. neoformans being a transient resident, commensal, or incitant of benign minimal lesions in the tracheobronchial tree is discussed. A comprehensive laboratory and clinical follow-up is warranted in patients from whose sputum or bronchial aspirate C. neoformans may be cultured even though definitive signs of cryptococcosis may be lacking.
A new medium composed of Tween 80, oxgall, caffeic acid, and Davis agar (TOC) that provides for the rapid presumptive identification of Candida albicans and Cryptococcus neoformans is described herein. C. albicans is differentiated from other yeasts by the sequential production of germ tubes and chlamydospores. In a comparison with cormeal agar control plates, there was an increase of chlamydospore-forming strains of C. albicans (97.1% versus 87.2%) and a decrease in the time required for chlamydospore formation (24 h versus 48 h). C. neoformans produced a brown pigment of TOC, which is specific for its identification, thus differentiating it from the other yeasts. A comparison of 24-h pigment production by C. neoformans on TOC with that of birdseed agar showed a dark, coffee brown color in the former cultures and a light brown color in the latter. The change in pigmentation of C. neoformans, as well as morphological changes in C. albicans, can be induced within 3 to 12 h and in not more than 24 h on the TOC medium.
Cryptococcus neoformans was cultured on one occasion from the oropharyngeal washings in 1 of 820 and from the interdigital areas of the feet in 6 of 723 healthy persons. Among the positive persons were two school children, two laboratory workers, one gardener, one plumber, and one printing press worker. Repeat cultures were negative in all seven positive persons; serological tests for cryptococcal antigen/antibody were negative in three. The results demonstrate that C. neoformans may occur as a transient inhabitant of the oropharynx or skin of healthy people.
An evaluation of a rapid caffeic acid-ferric citrate paper disk test for the identification of Cryptococcus neoformans, using 474 isolates of yeasts and yeastlike organisms, showed that 96.6, 97.7, and 98.3% of 176 isolates of C. neoformans produced brown to dark-brown pigment on disks incubated for 6 h at room temperature, 30 degrees C, and 37 degrees C, respectively. All C. neoformans produced brown to dark-brown pigment within 24 h. However, nonspecific pigmentation was encountered at all temperatures of incubation with one isolate of Trichosporon cutaneum and, at room temperature only, with one isolate of C. luteolus after 6 h. Other genera of yeasts produced similar pigmentation after 24 h at all temperatures. The age of the cultures tested or the types of media used before testing did not significantly affect the ability of C. neoformans to produce pigmentation at 37 degrees C. A positive test may prove useful for presumptive identification of C. neoformans, but a negative reaction should not be used to rule out an identification of this yeast. Since a number of false-negative and false-positive tests occur, it is necessary to confirm, by other biochemical tests, the identification of all organisms suspected of being C. neoformans, to reduct the serious risk of missing or misidentifying this important pathogen.
Cryptococcus neoformans was isolated repeatedly from a patient with epider-moid carcinoma and pulmonary blastomycosis. The isolate was atypical in that it had only a minute capsule, caused persistent infection but no perceptible disease in mice, and initially appeared not to assimilate trehalose. Only after an incubation of 2 to 3 weeks did utilization of this substrate become apparent.
A simplified Guizotia abyssinica seed medium, eliminating glucose, creatinine, and phosphate, was evaluated for the isolation and presumptive identification of Cryptococcus neoformans. Of 80 isolates of C. neoformans tested, 69 (86%) developed the characteristic brown pigment within 12 h on this medium as against only 5 isolates (6%) on the complete medium. In primary cultures of experimentally seeded specimens of sputum, bronchial aspirate, soil, and pigeon excreta, C. neoformans was recognizable within 3 to 5 days on the modified medium in contrast to the 3 to 8 days required on the complete medium. The results demonstrated that the simplified G. abyssinica seed agar, with or without diphenyl, is superior to the complete medium for the rapid development of the brown pigment by C. neoformans.
A cDNA encoding Cryptococcus neoformans orotidine monophosphate pyrophosphorylase (OMPPase) has been isolated by complementation of the cognate Escherichia coli pyrE mutant. The cDNA was used as a probe to isolate a genomic DNA fragment encoding the OMPPase gene (URA5). By using electroporation for the introduction of plasmid DNA containing the URA5 gene, C. neoformans ura5 mutants could be transformed at low efficiency. Ura+ transformants obtained with supercoiled plasmids containing the URA5 gene showed marked mitotic instability and contained extrachromosomal URA5 sequences, suggesting limited ability to replicate within C. neoformans. Transformants obtained with linear DNA were of two classes: stable transformants with integrated URA5 sequences, and unstable transformants with extrachromosomal URA5 sequences.
The sensitivity of three methods for the rapid identification of Cryptococcus neoformans was compared. These were: direct microscopy of india ink preparations, acridine orange staining followed by fluorescence microscopy and detection of cryptococcal capsular polysaccharide antigen by latex agglutination. The overall limit of detection was 3.5 +/- 5.4 X 10(3) CFU/ml (mean +/- SD, n = 27). When different strains were studied, no single method was consistently superior. False positive results were rare (two of 162 observations, 1.2%) but there were eight false negatives (4.9%), five of which were with acridine orange. Tests such as these are an invaluable aid in the rapid diagnosis of cryptococcal meningitis but they cannot be relied upon to detect low grade infections.