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A rapid simplified screening method that selectively detected the urease activity of 99.6% of 286 isolates of Cryptococcus neoformans within 15 min was developed for use in clinical microbiology laboratories.
Species of Filobasidiella, the agents of cryptococcosis, produced melanin-like pigments within 4 to 48 h with diphenol, aminophenol, and diaminobenzene compounds as substrates. The rate of phenyloxidase activity was found to be regulated by glucose and nitrogen catabolite repression. Increased glucose concentration reduced pigmentation of all serotypes of Filobasidiella, whereas repression by nitrogen sources varied with the strain. Glutamine repressed the phenyloxidases of all isolates except those of serotype B, and (NH4)2SO4 repressed the phenyloxidase of all isolates except that of serotype A. Tyrosine and glycine appeared to be near optimal for phenyloxidase activity but not necessarily for growth of all strain examined. Representatives of serotype C were unique in that their phenyloxidase system was adpative in contrast to the constitutive system found in the other serotypes. No single medium was found to support pigmentation of all strains of Cryptococcus neoformans within a 72-h incubation period; false-negative reactions can occur.
Results obtained with 433 yeast isolates indicated that the CN screen medium could be used with confidence for presumptive identification of Cryptococcus neoformans. Of 49 C. neoformans isolates tested, only four stock isolates yielded false-negative results upon initial testing. After repeated subculturing on Sabouraud agar and retesting, these four isolates yielded correct results. Essentially, no false-positive results were obtained, and the data suggested that false-negative results could be eliminated by using fresh isolates of C. neoformans preincubated at 25 degrees C on Sabouraud glucose agar.
A simple medium was developed, using esculin as the substrate, for the isolation and identification of Cryptococcus neoformans. C. neoformans produced a brown-black pigment on the medium; all other yeasts produced no pigment or were light yellow. Esculin is beta-glucose-6,7-dihydroxycoumarin. C. neoformans produced pigment because the 6,7-dihydroxycoumarin component of the esculin molecule was converted to a melanin-like pigment. We think the reaction was similar to the conversion of diphenols, aminophenols, and diaminobenzenes to melanin. Laboratory studies with isolates of C. neoformans, C. albidus, C. luteolus, and C. terreus and representatives of the genera Candida, Torulopsis, Geotrichum, and Rhodotorula, plus environmental field studies, demonstrated that over 95% of C. neoformans isolates were correctly identified, whereas all other fungi were excluded. Esculin agar was a sensitive, specific medium for the isolation and identification of C. neoformans. It was inexpensive and had a long storage life.
The recognition of Cryptococcus neoformans in Gram-stained smears of purulent exudates may be hampered by the presence of the large gelatinous capsule which apparently prevents definitive staining of the yeast-like cells. In such stained preparations, C. neoformans may appear either as round cells with gram-positive granular inclusions impressed upon a pale lavender cytoplasmic background or as gram-negative lipoid bodies.
An examination of 21 strains of Cryptococcus neoformans isolated from environmental and clinical sources in Germany revealed only serotypes A, D, and AD. Of these, 13 isolates were serotype A, 5 isolates were serotype D, and 3 isolates were identified as serotype AD. The absence of serotypes B or C confirms earlier reports from other European countries.
Four serotypes of Cryptococcus neoformans designated A, B, C, and D are currently recognized. Although an agglutination test is most often used to serotype C. neoformans in cultures, this test is not appropriate for typing the fungus in fixed tissues. A study to prepare fluorescent-antibody reagents for typing C. neoformans in cultures and to determine whether they can be used to type this fungus in fixed tissues was carried out. Antisera to one strain belonging to each of the four serotypes were prepared in rabbits by intravenous injection of whole Formalin-killed cryptococci. Each antiserum was labeled with fluorescein isothiocyanate and then adsorbed with cells of each of the heterologous serotypes. The adsorbed conjugates were then tested against six serotype A isolates and five isolates of each of the other three serotypes. Labeled serotype A or D antiserum adsorbed with either B or C cells stained the A and D, but not the B or C, isolates. Labeled serotype B antiserum adsorbed with A cells stained the B and C, but not the A or D, isolates. Labeled A antiserum absorbed with D cells differentiated A from D; labeled C antiserum absorbed with B cells differentiated C from B. Of the 21 test isolates, 17 could be serotyped in paraffin sections of tissues of experimentally infected mice.
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.
We have determined the in vitro susceptibilities of 57 strains of Cryptococcus gattii to nine antifungal agents and have compared the MICs for these strains with those for C. neoformans. MICs were determined by a microdilution reference method. Albaconazole and ravuconazole (MICs of 0.04 and 0.05 microg/ml, respectively) showed the best activities. Micafungin showed no activity (MIC of >128 microg/ml). In general, C. gattii was less susceptible than C. neoformans to all drugs tested, with the exception of amphotericin B and flucytosine.