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ABSTRACTWe describe a case of invasive fungal infection caused by Volvariella volvaceafollowing double umbilical cord blood transplantation (UCBT). Although infections caused by several mushroom species have been documented, we believe this to be the first published report of invasive infection with Volvariella volvacea, an edible mushroom belonging to Agaricales.

ABSTRACTWe analyzed 71 clinical and environmental Cryptococcus gattiistrains that had been isolated before or after the advent of azole antifungals to determine their level of heteroresistance to fluconazole (LHF). All strains of C. gattiimanifested heteroresistance, with LHFs that ranged between 4 μg/ml and 32 μg/ml. A considerably higher proportion of the C. gattiistrains (86%) than Cryptococcus neoformansstrains (46%) exhibited LHFs that were ≥16 μg/ml. No significant correlation was observed between the molecular type or serotypes of strains and their respective LHF. The strains which expressed a higher LHF were also more resistant to xenobiotics than the strains with a low LHF, and the level of resistance to xenobiotics was significantly higher than that reported for C. neoformans. The heteroresistant subpopulation, whose level of drug resistance had been raised in a stepwise manner to 64 μg/ml, reverted to the original LHF upon daily transfers in drug-free medium. Importantly, the strains with high LHFs were significantly more virulent than those with low LHFs. Since all the clinical isolates that had not been exposed to azole drugs as well as the environmental strains manifested heteroresistance to fluconazole, heteroresistance of C. gattiito azoles is an intrinsic mechanism as in C. neoformansand is associated with the strain's virulence.

ABSTRACTA recent report on several cases of invasive aspergillosis caused by Neosartorya udagawaesuggested distinctive patterns of disease progression between N. udagawaeand Aspergillus fumigatus. This prompted us to characterize N. udagawaein comparison to A. fumigatus. Our findings showed that both species exist in two mating types at similar ratios and produce gliotoxin. However, the thermotolerance of the two species differs: while A. fumigatusis able to grow at 55°C but not at 10°C, N. udagawaeis able to grow at 10°C but fails to grow at >42°C. Furthermore, compared to A. fumigatus, the conidia of N. udagawaerequire longer incubation periods to germinate at 37°C and are more susceptible to neutrophil attack as well as hydrogen peroxide; N. udagawaeis also less virulent in gp91phox-/-mice. These findings suggest that growth and susceptibility to the host response might account for the reduced virulence of N. udagawaeand the subtle distinction in the progression of the disease caused by the two species.

A recent report on several cases of invasive aspergillosis caused by Neosartorya udagawae suggested distinctive patterns of disease progression between N. udagawae and Aspergillus fumigatus. This prompted us to characterize N. udagawae in comparison to A. fumigatus. Our findings showed that both species exist in two mating types at similar ratios and produce gliotoxin. However, the thermotolerance of the two species differs: while A. fumigatus is able to grow at 55°C but not at 10°C, N. udagawae is able to grow at 10°C but fails to grow at >42°C. Furthermore, compared to A. fumigatus, the conidia of N. udagawae require longer incubation periods to germinate at 37°C and are more susceptible to neutrophil attack as well as hydrogen peroxide; N. udagawae is also less virulent in gp91phox–/–mice. These findings suggest that growth and susceptibility to the host response might account for the reduced virulence of N. udagawae and the subtle distinction in the progression of the disease caused by the two species.

ABSTRACTThe aim of this investigation was to study the effect of polysaccharide capsule on the gene expression in dendritic cells (DC) during their interaction with Cryptococcus neoformans. To this end, we used an encapsulated virulent strain of C. neoformansand a cap59gene-disrupted acapsular avirulent strain derived from the same genetic background. DC were exposed to encapsulated and acapsular C. neoformansstrains for 4 h and 18 h, and their transcriptional profiles were analyzed using the Affymetrix mouse gene chip U74Av2. A large number of DC genes were up-regulated after treatment with the acapsular strain. In particular, we observed the up-regulation of the genes involved in DC maturation, such as cell surface receptors, cytokines, and chemokines (interleukin-12 [IL-12], IL-2, IL-1α, IL-1β, IL-6, IL-10, tumor necrosis factor alpha, CCR7, CCL17, CCL22, CCL3, CCL4, CCL7, and CXCL10), membrane proteins, and the genes involved in antigen processing and presentation as well as cell cycle or apoptosis. The chemokine gene expression data were confirmed by real-time reverse transcription-PCR, while the expression of cytokine genes was correlated with their secretion. A completely different pattern of gene expression was observed for DC treated with an encapsulated strain of C. neoformans. In particular, no significant induction was observed in the expression of the genes mentioned above. Moreover, a number of genes, such as those coding for chemokines, were down-regulated. These results suggest that the polysaccharide capsule shrouding the cell wall of C. neoformansplays a fundamental role in inducing DC response, highlighting the molecular basis of the true nature of immune silencing exerted by capsular material.

The aim of this investigation was to study the effect of polysaccharide capsule on the gene expression in dendritic cells (DC) during their interaction with Cryptococcus neoformans. To this end, we used an encapsulated virulent strain of C. neoformans and a cap59 gene-disrupted acapsular avirulent strain derived from the same genetic background. DC were exposed to encapsulated and acapsular C. neoformans strains for 4 h and 18 h, and their transcriptional profiles were analyzed using the Affymetrix mouse gene chip U74Av2. A large number of DC genes were up-regulated after treatment with the acapsular strain. In particular, we observed the up-regulation of the genes involved in DC maturation, such as cell surface receptors, cytokines, and chemokines (interleukin-12 [IL-12], IL-2, IL-1, IL-1β, IL-6, IL-10, tumor necrosis factor alpha, CCR7, CCL17, CCL22, CCL3, CCL4, CCL7, and CXCL10), membrane proteins, and the genes involved in antigen processing and presentation as well as cell cycle or apoptosis. The chemokine gene expression data were confirmed by real-time reverse transcription-PCR, while the expression of cytokine genes was correlated with their secretion. A completely different pattern of gene expression was observed for DC treated with an encapsulated strain of C. neoformans. In particular, no significant induction was observed in the expression of the genes mentioned above. Moreover, a number of genes, such as those coding for chemokines, were down-regulated. These results suggest that the polysaccharide capsule shrouding the cell wall of C. neoformans plays a fundamental role in inducing DC response, highlighting the molecular basis of the true nature of immune silencing exerted by capsular material.

ABSTRACTThe polysaccharide capsule is known to be the major factor required for the virulence of Cryptococcus neoformans. We have cloned and characterized a gene, designated CPS1, that encodes a protein containing a glycosyltransferase moiety and shares similarity with the type 3 polysaccharide synthase encoded by the cap3Bgene of Streptococcus pneumoniae. Cps1p also shares similarity with hyaluronan synthase of higher eukaryotes. Deletion of the CPS1gene from a serotype D strain of C. neoformansresulted in a slight reduction of the capsule size as observed by using an India ink preparation. The growth at 37°C was impaired, and the ability to associate with human brain endothelial cells in vitro was also significantly reduced by the deletion of CPS1. Using site-specific mutagenesis, we showed that the conserved glycosyltransferase domains are critical for the ability of the strain to grow at elevated temperatures. A hyaluronan enzyme-linked immunosorbent assay method demonstrated that CPS1is important for the synthesis of hyaluronan or its related polysaccharides in C. neoformans. Comparisons between the wild-type and the cps1Δstrains, using three different transmission electron microscopic methods, indicated that the CPS1gene product is involved in the composition or maintenance of an electron-dense layer between the outer cell wall and the capsule. These and the virulence studies in a mouse model suggested that the CPS1gene is important in the pathobiology of C. neoformans.

A 47-year-old diabetic man with chronic renal failure presented with a 1-month history of complete ptosis of the left upper eyelid, left proptosis, and left-sided headache. During the course of the patient’s care, other significant diagnoses were excluded, such as orbital inflammatory syndrome, carotid-cavernous syndrome, and cavernous sinus thrombosis. Neuroimaging revealed only minimal left sphenoid sinus disease. Sphenoid biopsy revealed the presence of septate hyphae on Gram staining and produced a fungal culture characteristic of Schizophyllum commune. Minimal sphenoid sinus infection in a patient with chronic medical issues and probable immunosuppression predisposed this patient to fungal rhino-orbital infection. Several weeks of intravenous liposomal amphotericin treatment on an outpatient basis yielded resolution of clinical symptoms.

Several genes are essential for Cryptococcus neoformans capsule synthesis, but their functions are unknown. We examined the localization of glucuronoxylomannan (GXM) in strain B-3501 and in cap59 mutants B-4131 and C536. Wild-type strain B-3501 showed a visible capsule by India ink staining and immunofluorescence with anticapsular monoclonal antibodies (MAbs) 12A1 and 18B7. B-4131, a mutant containing a missense mutation in CAP59, showed no capsule by India ink staining but revealed the presence of capsular polysaccharide on the cell surface by immunofluorescence. The cap59 gene deletion mutant (C536), however, did not show a capsule by either India ink staining or immunofluorescence. Analysis of cell lysates for GXM by enzyme-linked immunosorbent assay revealed GXM in C536 samples. Furthermore, the epitopes recognized by MAbs 12A1, 2D10, 13F1, and 18B7 were each detected in the cytoplasm of all strains by immunogold electron microscopy, although there were differences in location consistent with differences in epitope synthesis and/or transport. In addition, the cells of B-3501 and B-4131, but not those of the cap59 deletant, assimilated raffinose or urea. Hence, the missense mutation of CAP59 in B-4131 partially hampered the trafficking of GXM but allowed the secretion of enzymes involved in hydrolysis of raffinose or urea. Furthermore, the cell diameter and volume for strain C536 are higher than those for strain B-3501 or B-4131 and may suggest the accumulation of cellular material in the cytoplasm. Our results suggest that CAP59 is involved in capsule synthesis by participating in the process of GXM (polysaccharide) export.

ABSTRACTSeveral genes are essential for Cryptococcus neoformanscapsule synthesis, but their functions are unknown. We examined the localization of glucuronoxylomannan (GXM) in strain B-3501 and in cap59mutants B-4131 and C536. Wild-type strain B-3501 showed a visible capsule by India ink staining and immunofluorescence with anticapsular monoclonal antibodies (MAbs) 12A1 and 18B7. B-4131, a mutant containing a missense mutation in CAP59, showed no capsule by India ink staining but revealed the presence of capsular polysaccharide on the cell surface by immunofluorescence. The cap59gene deletion mutant (C536), however, did not show a capsule by either India ink staining or immunofluorescence. Analysis of cell lysates for GXM by enzyme-linked immunosorbent assay revealed GXM in C536 samples. Furthermore, the epitopes recognized by MAbs 12A1, 2D10, 13F1, and 18B7 were each detected in the cytoplasm of all strains by immunogold electron microscopy, although there were differences in location consistent with differences in epitope synthesis and/or transport. In addition, the cells of B-3501 and B-4131, but not those of the cap59deletant, assimilated raffinose or urea. Hence, the missense mutation of CAP59in B-4131 partially hampered the trafficking of GXM but allowed the secretion of enzymes involved in hydrolysis of raffinose or urea. Furthermore, the cell diameter and volume for strain C536 are higher than those for strain B-3501 or B-4131 and may suggest the accumulation of cellular material in the cytoplasm. Our results suggest that CAP59is involved in capsule synthesis by participating in the process of GXM (polysaccharide) export.

Cryptococcus neoformans is the etiologic agent of cryptococcosis. Two mating types exist in this fungus, MAT alpha and MATa. The CPRa gene of C. neoformans is a MATa strain-specific gene and encodes a putative seven-transmembrane domain pheromone receptor. Unlike the other reported fungal pheromone receptors, CPRa shows functional diversity. Deletion of CPRa drastically affects mating efficiency but does not abolish mating. CPRa expression is developmentally regulated and is not affected by deletion of the transcriptional regulator STE12a. The expression of CPRa is markedly increased by shifting cultures from liquid to solid media. CPRa also plays a significant role in virulence. Delta cpra cells produce smaller capsules in the brains of mice than the wild-type cells, and the mice infected with Delta cpra survive significantly longer than those receiving the wild-type strain. Our results suggest that the MATa pheromone receptor of C. neoformans is not only required for mating but also important for survival and growth of the fungus in host tissue.

ABSTRACTCryptococcus neoformansis the etiologic agent of cryptococcosis. Two mating types exist in this fungus, MATα and MATa. The CPRagene of C. neoformansis a MATastrain-specific gene and encodes a putative seven-transmembrane domain pheromone receptor. Unlike the other reported fungal pheromone receptors, CPRashows functional diversity. Deletion of CPRadrastically affects mating efficiency but does not abolish mating. CPRaexpression is developmentally regulated and is not affected by deletion of the transcriptional regulator STE12a. The expression of CPRais markedly increased by shifting cultures from liquid to solid media. CPRaalso plays a significant role in virulence. Δcpracells produce smaller capsules in the brains of mice than the wild-type cells, and the mice infected with Δcprasurvive significantly longer than those receiving the wild-type strain. Our results suggest that the MATapheromone receptor of C. neoformansis not only required for mating but also important for survival and growth of the fungus in host tissue.

Chain lengths and cyclization patterns of microbial polyketides are generally determined by polyketide synthases alone. Fungal polyketide melanins are often derived from a pentaketide 1,8-dihydroxynaphthalene, and pentaketide synthases are used for synthesis of the upstream pentaketide precursor, 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN). However, Aspergillus fumigatus, a human fungal pathogen, uses a heptaketide synthase (Alb1p) to synthesize its conidial pigment through a pentaketide pathway similar to that which produces 1,8-dihydroxynaphthalene-melanin. In this study we demonstrate that a novel protein, Ayg1p, is involved in the formation of 1,3,6,8-THN by chain-length shortening of a heptaketide precursor in A. fumigatus. Deletion of the ayg1 gene prevented the accumulation of 1,3,6,8-THN suggesting the involvement of ayg1 in 1,3,6,8-THN production. Genetic analyses of double-gene deletants suggested that Ayg1p catalyzes a novel biosynthetic step downstream of Alb1p and upstream of Arp2p (1,3,6,8-THN reductase). Further genetic and biochemical analyses of the reconstituted strains carrying alb1, ayg1, or alb1 + ayg1 indicated that Ayg1p is essential for synthesis of 1,3,6,8-THN in addition to Alb1p. Cell-free enzyme assays, using the crude Ayg1p protein extract, revealed that Ayg1p enzymatically shortened the heptaketide product of Alb1p to 1,3,6,8-THN. Thus, the protein Ayg1p facilitates the participation of a heptaketide synthase in a pentaketide pathway via a novel polyketide-shortening mechanism in A. fumigatus.

The incidence of cryptococcosis rose dramatically with the advent of the acquired immune deficiency syndrome (AIDS) epidemic in the early 1980s until the early 1990s. The frequency of cryptococcosis has been declining since mid 1990s in Europe and America due to the development of more effective antiretroviral therapy and prophylactic treatment regimens designed to prevent fungal infections. The disease, however, is still recognized as one of the most common life-threatening opportunistic fungal infections in immunocompromised patients, particularly among those infected with human immunodeficiency virus (HIV). For this reason, research interest in clinical and biological aspects of the disease remains high. In addition to previously embarked areas of research, the cryptococcal research community has taken advantage of the current sequencing technology and initiated genome sequencing of Cryptococcus neoformans var. neoformans. This review includes various areas of research interest ranging from pathobiology, biochemistry and immunology, to genomics.

A transformation system using resistance to the antibiotic cycloheximide as a dominant selectable marker was developed for the pathogenic yeast Cryptococcus neoformans. A 3.5 kb DNA fragment containing a gene encoding the ribosomal protein L41 was cloned from a wild-type strain of C. neoformans which is sensitive to cycloheximide. The open reading frame of the L41 gene contains five introns and encodes a protein of 107 amino acids, which is similar to those reported for other yeasts. The cycloheximide resistance gene to be used as a marker was constructed by replacing a DNA segment of the wild-type L41 gene, which contained the amino acid proline at its 56th position with a homologous DNA segment from a mutant strain resistant to cycloheximide that contained leucine in that position. Cycloheximide resistant transformants were obtained by electroporation on YEPD plates, supplemented with 10–20 µg/ml cycloheximide, at a maximum efficiency of 300 transformants/µg plasmid DNA. While with other genes, most transformants of serotype D in C. neoformans maintain the transforming DNA as episomes, the cycloheximide-resistant transformants were all the result of ectopic genomic integration events. Copyright © 2000 John Wiley & Sons, Ltd.

A transformation system using resistance to the antibiotic cycloheximide as a dominant selectable marker was developed for the pathogenic yeast Cryptococcus neoformans. A 3.5 kb DNA fragment containing a gene encoding the ribosomal protein L41 was cloned from a wild‐type strain of C. neoformanswhich is sensitive to cycloheximide. The open reading frame of the L41gene contains five introns and encodes a protein of 107 amino acids, which is similar to those reported for other yeasts. The cycloheximide resistance gene to be used as a marker was constructed by replacing a DNA segment of the wild‐type L41gene, which contained the amino acid proline at its 56th position with a homologous DNA segment from a mutant strain resistant to cycloheximide that contained leucine in that position. Cycloheximide resistant transformants were obtained by electroporation on YEPD plates, supplemented with 10–20 µg/ml cycloheximide, at a maximum efficiency of 300 transformants/µg plasmid DNA. While with other genes, most transformants of serotype D in C. neoformansmaintain the transforming DNA as episomes, the cycloheximide‐resistant transformants were all the result of ectopic genomic integration events. Copyright © 2000 John Wiley & Sons, Ltd.

ABSTRACTCryptococcus neoformansisolates that exhibited unusual patterns of resistance to fluconazole and voriconazole were isolated from seven isolates from two different geographical regions: one isolate from an Israeli non-AIDS patient and six serial isolates from an Italian AIDS patient who had suffered six recurrent episodes of cryptococcal meningitis. Each isolate produced cultures with heterogeneous compositions in which most of the cells were susceptible, but cells highly resistant to fluconazole (MICs, ≥64 μg/ml) were recovered at a variable frequency (7 × 10−3to 4.6 × 10−2). Evidence showed that this type of resistance is innate and is unrelated to drug exposure since the Israeli patient had never been treated with azoles or any other antimycotic agents. Analysis of clonal subpopulations of these two strains showed that they exhibited heterogeneous patterns of resistance. The number of subpopulations which grew on fluconazole or voriconazole agar declined progressively with increasing azole concentration without a sharp cutoff point. For the Italian serial isolates, the number of clonal populations resistant to fluconazole (64 μg/ml) and voriconazole (1 μg/ml) increased steadily, yielding the highest number for the isolate from the last episode. Attempts to purify a sensitive subpopulation failed, but clones highly resistant to fluconazole (100 μg/ml) and moderately resistant to voriconazole (1 μg/ml) always produced a homogeneous population of resistant cells. Upon maintenance on drug-free medium, however, the majority of the homogeneously resistant cells of these subclones lost their resistance and returned to the stable initial heteroresistant phenotype. The pattern of heteroresistance was not affected by the pH or osmolarity of the medium but was influenced by temperature. The resistance appeared to be suppressed at 35°C and was completely abolished at 40°C. Although heterogeneity in azole resistance among subpopulations of single isolates has been reported for Candidaspecies, the transient changes in expression of resistance under different growth conditions reported here have not been observed in fungal pathogens.

The extracellular polysaccharide capsule produced by Cryptococcus neoformans is essential for its pathogenicity. We have isolated and characterized a gene, (AP64, which is required for capsule formation. An encapsulated strain created by complementation of the cap64 mutation produced fatal infection of mice within 25 days, while the cap64 acapsular strain was avirulent. Gene deletion of CAP64 from a wild-type strain resulted in the loss of capsule as well as virulence. Contour-clamped homogeneous electric field gel analysis indicates that CAP64 is located on chromosome III which is different from the localization of another capsule-related gene, CAP59. The nonlinkage between CAP64 and CAP59 was also supported by classical recombinational analysis. Database searches did not reveal any sequence with high similarity to CAP64. We also found that the CAP64 locus is contiguous to a convergently transcribed gene which has significant similarity to the gene encoding the yeast proteasome subunit, PRE1. The distance between the cDNA ends of these two genes is only 22 bp. This study confirms the previous molecular genetic evidence that capsule is an essential factor for the virulence of C. neoformans in the murine model.

Electron microscopy of Filobasidiella neoformans, the perfect state of Cryptococcus neoformans, revealed basidiomycete doliporesepta between hyphal cells and also between clamp connections and adjacent cells. The pore-occluding material was a heterogeneous flattened plate with dark margins and a lighter center, as seen in the species of Filobasidium. Representative basidiomycete parenthesomes were lacking, and endoplasmic reticulum was seen in the dolipore region.

Abstract: We report the generation of stable plasmids constructed by inserting specific DNA sequences into previously known unstable vectors. These sequences were obtained from a DNA library recovered from a previously reported stable minichromosome created by electroporative transformation in Cryptococcus neoformans (Varma and Kwon-Chung 1994). A 6-kb insert from this minichromosome significantly enhanced both the frequencies at which URA5 transformants were obtained as well as the stability of their uracil prototrophy on non-selective media. A 1.5-kb sequence of this insert contained telomeric sequence repeats which when introduced into plasmids resulted in significant increases in transformation frequency. A 1081-bp sequence (STAB), present in the remainder of the insert, had an ARS-like function enhancing the episomal maintenance of plasmids in the transformants regardless of the gene (ADE2/URA5) used as a selection marker.

Aspergillus quadrilineatus was found to be the etiologic agent of pansinusitis in a patient suffering from acute nonlymphoblastic leukemia and who had undergone allogeneic bone marrow transplantation. A. quadrilineatus was cultured from biopsy specimens of the maxillary sinus, and tissue sections with fungal stains showed a necrotic area containing dichotomously branching septate hyphae, which is morphologically consistent with Aspergillus species. The patient was successfully treated with a combination of surgical debridement, granulocyte transfusions, and intravenous administration of amphotericin B-cholesterol sulfate colloidal dispersion. This is the first report of an infection caused by A. quadrilineatus.

A 7-kb linear plasmid, harbored by a URA5 transformant, hybridized to all the chromosomes of Cryptococcus neoformans separated by contour-clamped homogeneous electric field electrophoresis. Its linear maintenance was determined to have been facilitated by the presence of telomere-like sequences at its free ends. Hybridization of this plasmid to AccI-digested genomic DNAs of 26 C. neoformans strains generated 21 unique DNA fingerprints. The DNA fingerprints of isolates within the same serotype were more similar to one another than to those from different serotypes. An acapsular clinical isolate, strain 602, widely used in immunological studies and previously thought to be in serotype D, showed DNA fingerprints typical of serotype A isolates. Isogenic strains of C. neoformans exhibited DNA fingerprints that were identical to one another. The DNA fingerprints were stable and reproducible in spite of repeated transfers in the laboratory on either complex (1% yeast extract, 2% Bacto Peptone, 2% glucose) or minimal (yeast nitrogen base) medium. The DNA fingerprints of isolates recovered from primary blood and cerebrospinal fluid cultures of patients for whom AIDS had been diagnosed showed that the original infection in each of these patients contained a homogeneous population of C. neoformans. The DNA fingerprints of isolates recovered from different tissues of infected mice and from patients undergoing different drug therapy regimens were also found to be very stable.

Protoplasts of Cryptococcus neoformans contain phenoloxidase as a membrane-bound enzyme. The enzyme appeared to be attached on the inner side of cytoplasmic membranes. Synthesis of the enzyme was derepressed by low levels of glucose but was not affected by the level of ammonium. Copper chelators which inhibited the phenoloxidase of other organisms did not affect cryptococcal enzymes. However, cyanide- or iron-chelating agents such as hydroximide derivates or 8-hydroxyquinoline were effective inhibitors, suggesting that cryptococcal phenoloxidase is an iron-containing enzyme. Phenoloxidase of C. neoformans catalyzed the oxidation of various diphenols via dopachrome and labile intermediates to melanin polymers. The kinetic constants (Km) of the phenoloxidase and the permease for dopamine and norepinephrine were low. The correlation between phenoloxidase and the preferential growth of C. neoformans in the host brain is discussed.

Analysis of the karyotypes of multiple Candida albicans isolates by pulsed-field electrophoresis confirms the observation by Lasker et al. of eight chromosomes. The genes previously assigned to chromosome 1 in fact fall into two groups, one (including ADE1, SOR9, and CDC10) is linked to the ribosomal DNA genes on a chromosome called R, whereas the others are found on chromosome 1. Chromosome R varies in electrophoretic mobility among strains, usually running equal to or faster than chromosome 1 but in rare cases running slower than chromosome 1. In strain 1012A, the decreased mobility of one homolog is associated with the very large majority of the rDNA genes being on that homolog; the second homolog, with only a few copies, migrates with chromosome 2. Linkage analysis by using spheroplast fusion confirms the gene assignments made by hybridization to blots of the electrophoretic karyotype. A newly cloned gene, LYS2, hybridizes to chromosome 1.

When type I Candida stellatoidea is plated onto sucrose agar at levels in excess of 10(8) cells, some isolates spontaneously form sucrose-positive colonies. These isolates do not display typical type I phenotypes but instead exhibit phenotypes intermediate between type I C. stellatoidea and C. albicans. Also, this phenotypic change only occurs in conjunction with a chromosomal rearrangement. These rearrangements have been studied in a strain naturally marked for methionine auxotrophy. Chromosome-size DNA bands separated by pulsed-field gel electrophoresis were probed with genes cloned from C. albicans. The hybridization pattern indicated that the genes on several chromosomes underwent extensive rearrangement.

• A zygomycotic (phycomycotic) orbital cellulitis developed in a healthy 9-year-old boy. Because of the involved tissue's unfamiliar histopathologic features, an initial diagnosis of eosinophilic granuloma was made and appropriate treatment was delayed for more than six months. The patient's slowly progressive form of zygomycosis was clinically and morphologically similar to that described in previously recorded cases. All three cases occurred in otherwise healthy children living within the United States. Their involved tissues had mixed histopathologic features of chronic granulomatous entomophthoramycosis and acute necrotizing mucormycosis; however, unlike entomophthoramycosis, the fungi in these cases may invade the walls of blood vessels and cause severe tissue necrosis. We believe that these three cases represent a distinct clinicopathologic variant of facial-cranial zygomycosis not previously delineated in the literature. Although they are aggressive, these infections are not as fulminant as in classic mucormycosis, but can nevertheless be lethal.

Double-stranded RNA viruses were detected in a strain of Blastomyces dermatitidis isolated from a patient in Uganda. The viral particles are spherical (mostly 44 to 50 nm in diameter) and consist of about 25% double-stranded RNA (5 kb) and 75% protein (90 kDa). The virus contains transcriptional RNA polymerase activity; it synthesized single-stranded RNA in vitro in a conservative manner. The newly synthesized single-stranded RNA was a full-length strand, and the rate of chain elongation was approximately 170 nucleotides per min. The virus-containing strain shows no morphological difference from virus-free strains in the mycelial phase. Although the association with the presence of the virus is unclear, the virus-infected strain converts to the yeast form at 37 degrees C, but the yeast cells fail to multiply at that temperature.

A naturally occurring Mel- variant of Cryptococcus neoformans was isolated from the wild type. The effect of phenoloxidase activity on virulence was analyzed on genetically constructed Mel+ and Mel- isolates. The traits Mel+ and virulence in mice, as measured by cumulative mortality and replication potential in brain tissue, cosegregated among the progeny of a Mel+ X Mel- cross. Revertants (MelR) isolated during the course of the cumulative mortality experiment were used to compare virulence in isogenic sets of Mel- and MelR. In two separate sets of such isolates, Mel+ phenotype and virulence coreverted. Measurement of substrate uptake and phenoloxidase activity showed that loss of detectable phenoloxidase was the basis for the Mel- phenotype and that enzyme activity reappeared in the MelR isolates. An intermediate phenotype, Melbg, was also described. Cosegregation and coreversion of the melanin phenotype and virulence suggest that phenoloxidase is a virulence factor in C. neoformans.

The relationship between extracellular proteinase and the virulence for mice in Candida albicans was studied by using a set of three isolates. The set included a proteinase-producing parent (C9), a proteinase-deficient mutant derived from C9 by nitrous acid treatment (C9M1), and a spontaneous revertant (C9M1M) obtained by mouse passage of C9M1. The morphological markers and the carbon assimilation pattern were identical in these isolates. Isolate C9 produced a high level of proteinase in vitro and caused fatal infection (100%) within 21 days. The mutant produced no detectable enzymes in vitro, and all mice survived until day 22. Only 30% of the mice infected with C9M1 died between day 23 and 30. The isolates recovered from the dead mice were found to be proteinase sufficient, indicating that the mice died after the organism in tissue had reverted. The C9M1M isolate produced proteinase in vitro at 44% the level of C9 and induced fatal infection in 90% of the mice within 30 days. The number of CFU recovered from the kidneys correlated with the level of proteinase produced in vitro and, in turn, the rate of fatal infection produced by the isolates. These results support a previous observation indicating that proteinase activity is one of the virulence factors associated with C. albicans.

AbstractThe genus Filobasidiellacontains two species, F. neoformansand F. depauperata.The 5S ribosomal RNA genes in the two species were found to have completely different genomic arrangements. We previously reported that the 5S rRNA gene of F. neoformans, the type species of genus Filobasidiella, is located 1 kb upstream from the 18S rRNA gene within the 8.6-kb HindIII fragment of the ribosomal DNA repeat unit. The present study demonstrates the 5S rRNA gene to be absent from the rDNA repeat in F. depauperata, but is dispersed in the genome. Analysis of the nucleotide sequence from five randomly isolated 5S rDNA clones of F. depauperatashowed heterogeneity at six different positions of the putative transcription region. The genus Filobasidiellais the first genus of Eumycota reported to have the 5S rRNA with two different genomic arrangements.

Four strains of Cryptococcus neoformans var. gattii originating from Eucalyptus camaldulensis, three from Australia and one from San Francisco, were tested for their serotype, virulence for mice, and a number of genetic and molecular characteristics. All were found to be serotype B and showed significantly higher virulence for mice than did the type strains of C. neoformans var. gattii and Filobasidiella neoformans var. bacillispora, which were obtained from human cryptococcosis cases. Electrophoretic karyotypes of the strains from Australia were identical, although they were collected from sites at least 15 to 500 km apart. The electrophoretic karyotype of the strain from San Francisco was the same as that of the Australian isolates except for the mobility of one chromosome. On the contrary, no two isolates of serotype B (of a total of 11) from clinical sources were the same, regardless of their geographic origin. Furthermore, none of the clinical isolates showed a chromosomal banding pattern identical to that of Eucalyptus-originated strains. The Eucalyptus-originated strains failed to form dikaryons when crossed with the tester strains of the two varieties of F. neoformans. Hybridization analysis with a nucleic acid probe (AccuProbe C. neoformans Culture Confirmation Test; Gen-Probe Inc., San Diego, Calif.), however, showed signals of equal intensity for clinical strains and the Eucalyptus-originated strains. Various fungi phylogenetically related to C. neoformans, including a phenol oxidase-positive strain of Cryptococcus laurentii obtained from E. camaldulensis, were negative in the nucleic acid hybridization test. These observations confirm that, in spite of karyotypic differences and the lack of dikaryon formation with the tester strains of F. neoformans, Eucalyptus-originated C. neoformans var. gattii is the same organism as those isolated from cases of human infection. Furthermore, the C. neoformans culture confirmation test using a commercial nucleic acid probe is specific for C. neoformans.

Cryptococcus neoformans var. neoformans ura5 mutants were transformed with linearized or circular plasmids containing the C. neoformans orotidine monophosphate pyrophosphorylase gene. Following electroporation, randomly isolated transformants were analyzed for the mitotic and meiotic stability of uracil prototrophy. All stable transformants tested showed nonspecific ectopic integration. Uracil prototrophy in these transformants was stable through meiosis. Some of the stable transformants showed integration of both URA5 and vector sequences, while others lacked any vector sequences. Unstable transformants exhibited the presence of an autonomously replicating plasmid which had undergone significant sequence rearrangement. The autonomously replicating plasmid in the transformants was observed to be the same size or smaller than the transforming plasmid, was maintained in a linear form, and had acquired a genomic sequence(s) with homology to a sequence(s) on all the chromosomes. The conservation of a 300-bp sequence at the 5' end of the URA5 gene was observed in all the rearranged plasmids. These results suggest mechanisms of plasmid maintenance in C. neoformans that are different from those reported for other yeasts. The ura5 mutant was significantly less virulent than the wild type. The transformants did not recover virulence regardless of prototrophic stability.

A pair of congenic Cryptococcus neoformans var. neoformans strains, B-4476 (a mating type) and B-4500 (alpha mating type), that presumably differ only in mating type was constructed. This pair and their progeny, five alpha type and five a type, were tested for virulence in mice. In the parent strains as well as the progeny, alpha type was clearly more virulent than a type. In addition, death tended to occur earlier among the alpha-strain-infected mice that died than among the mice that died by infection caused by a strains. These data strongly suggest the genetic association of virulence with mating type in this human fungal pathogen.

Candida stellatoidea is classically distinguished from C. albicans by the ability of the latter species to assimilate sucrose. We show here that sucrose-positive revertants of C. stellatoidea type II are readily isolated and that C. stellatoidea type II strains probably resulted from a mutation in the sucrase gene of C. albicans. The revertants were not laboratory contaminants, as determined by restriction fragment length polymorphism analysis and retention of an auxotrophic marker. The reversion of three tested strains was accompanied by 16 to 110-fold increases in expression of a sucrase/alpha-glucosidase but not an invertase, with a Km for sucrose of about 1 mM. The enzyme activity was assayable in intact cells. The drastically increased expression of such an enzyme would allow extracellular sucrose hydrolysis and assimilation of the monosaccharide products.

Mutant strains of Candida albicans were obtained by selecting for cells that escaped agglutination by a polyclonal antiserum raised against standard C. albicans serotype A isolate B311. Mutants were obtained from strains B311 and B792 and from four strains isolated from patients with acquired immunodeficiency syndrome. All 15 tested mutants retained characteristic sugar assimilation patterns. All but one of the mutants retained the ability to form germ tubes and chlamydospores. Two mutants from an acquired immunodeficiency syndrome-derived isolate were deficient in binding complement ligands iC3b and C3d, whereas another mutant was deficient in binding ligand iC3b but not C3d. The hyphae of these three mutants lacked antigens when examined by Western immunoblotting with monoclonal antibody Ca-A, which detects several glycoproteins, including C3d-binding proteins. One of the complement-binding-deficient mutants was tested for its ability to colonize the gastrointestinal tract of rabbits but did not differ from the wild-type parent in site or degree of colonization. The proton magnetic resonance spectra of bulk mannan carbohydrate extracted from tested mutants showed the loss of a signal characteristic of the mannosyl alpha-PO4 linkage; each mutant also had a distinct pattern of other changes.

Genetic similarities and differences between type I and type II Candida stellatoidea were studied. The electrophoretic karyotype, mitochondrial DNA (mtDNA) restriction patterns, and midrepeat sequence of nuclear DNA in type I C. stellatoidea were clearly distinguishable from those of a reference culture of Candida albicans. The karyotype and the major bands of the midrepeat sequence of type II C. stellatoidea were indistinguishable from those of the reference C. albicans. The mtDNA restriction patterns of four type I isolates were homogeneous regardless of the endonucleases and probes used. The mtDNA restriction patterns of type II C. stellatoidea varied from strain to strain. Some of them were identical to that of C. albicans, while others were the same as that of type I C. stellatoidea. Immunofluorescence with C. albicans serotype A-specific monoclonal antibody indicated that the four isolates of type I C. stellatoidea were serotype B (non-A), whereas all three type II isolates studied were serotype A. Taken together, these results support the hypothesis that the isolates of C. stellatoidea type II studied are sucrose-negative mutants of serotype A C. albicans. Since C. stellatoidea type I differs from C. albicans in several major genetic characteristics, it cannot be viewed as a simple mutant derived from C. albicans. Hybrids produced by protoplast fusion of type I and type II cells were capable of assimilating sucrose, indicating that the sucrose-negative phenotypes of the parents are due to different mutations.

Seven isolates of Candida stellatoidea were studied for their electrophoretic karyotype, virulence for mice, sensitivity to UV radiation, growth rate in vitro, reaction on cycloheximide-indicator medium, and proteinase activity. The isolates exhibited one of two distinct electrophoretic karyotypes as determined by orthogonal field alternating gel electrophoresis (OFAGE). Four isolates, including the type culture of C. stellatoidea, belonged to electrophoretic karyotype type I by OFAGE, showing eight to nine bands of which at least two bands were less than 1,000 kilobases in size as estimated by comparison with the DNA bands of Saccharomyces cerevisiae. These isolates failed to produce fatal infection in mice within 20 days when 5 X 10(5) cells were injected intravenously. The yeasts were cleared from the kidneys of two of three mice tested by day 30. Type I showed proteinase activity on bovine serum albumin agar at pH 3.8 and produced a negative reaction on cycloheximide-bromcresol green medium within 48 h. The three grouped in type II by OFAGE showed banding patterns similar to those of a well-characterized isolate of Candida albicans. The isolates of type II had an electrophoretic karyotype of six to seven bands approximately 1,200 kilobases or greater in size. All three type II isolates were highly virulent for mice, producing fatality curves similar to those of a previously studied C. albicans isolate. From 80 to 90% of the mice injected with 5 X 10(5) cells intravenously died within 20 days. The type II isolates produced a positive reaction on cycloheximide-bromcresol green agar and showed no proteinase activity on bovine serum albumin agar at the low pH. In addition, the type II isolates grew faster and were significantly more resistant to UV irradiation than the type I isolates. These results indicated that type II, but not type I, isolates can be considered simply as sucrose-negative C. albicans.

DNA fingerprint patterns of 156 Cryptococcus neoformans isolates (26 AIDS patients, 46 non-AIDS patients, and 40 environmental sources) from both varieties (126 C. neoformans var. neoformans and 30 C. neoformans var. gattii isolates) and from seven countries were analyzed by using the DNA probe UT-4p. Nine and twelve distinct DNA fingerprint patterns were observed for isolates of the C. neoformans var. neoformans and var. gattii, respectively. No pattern was unique to AIDS patients, non-AIDS patients, or the environment. Pattern II was observed more often in non-AIDS patients (8 of 23) than in AIDS patients (0 of 25). Pattern V was the most prevalent pattern (42 of 82) in clinical and environmental isolates. Isolates from three AIDS patients in Burundi and Zaire exhibited patterns identical to each other but different from those of isolates collected from their houses (i.e., dust of floors, walls, etc.) or a nearby pigeon coop. DNA fingerprint stability was determined for 53 isolates from nine non-AIDS patients at different time intervals during 5 to 128 weeks of antifungal therapy. For eight patients, the fingerprint pattern was stable while the ninth may have had a mixed infection. Pattern II was observed in 4 of 9 patients, which is similar to 4 of 14 in other non-AIDS patients as reported here. In spite of the extensive pattern heterogeneity among 15 C. neoformans var. gattii isolates in Australia, the patterns observed in seven California isolates were quite different from those in Australia. Among isolates of C. neoformans var. gattii, one fingerprint pattern (designated b) was observed in several countries of the Far East. The fingerprint patterns of two of three environmental isolates from Eucalyptus camaldulensis trees in Australia were identical to those of 2 of the 12 clinical isolates from the country.

Cryptococcus neoformans var. neoformans (74 isolates) and C. neoformans var. gattii (44 isolates) were used to test urease activity after growth on both yeast extract-glucose-peptone agar (YEPG) and on YEPG supplemented with 100 microM EDTA. Every isolate grown on YEPG agar for 48 h at 30 degrees C produced a positive reaction within 1 h in a modified rapid urease assay at 37 degrees C. However, isolates grown on YEPG with 100 microM EDTA showed a distinct pattern which corresponded to their varietal status. All but 1 of 74 C. neoformans var. neoformans isolates (98.7%) produced a positive reaction within 1 to 4 h, while none of 44 C. neoformans var. gattii isolates produced a positive reaction within the same period. The urease inhibition results and the canavanine-glycine-bromthymol blue agar test results showed 100% correlation among isolates of C. neoformans var. gattii and 98.7% correlation among isolates of C. neoformans var. neoformans. Two representative isolates of C. neoformans var. gattii (serotypes B and C) were further tested for urease during a prolonged incubation period in urea broth. These isolates failed to show a positive reaction even after 11 h of incubation. The uptake of EDTA was negligible in the two varieties. Extracts of cells grown on YEPA agar showed a high level of urease activity in both varieties. Extracts of cells grown on the agar with 100 microM EDTA showed a marked reduction (86%) of urease activity in one isolate of C. neoformans var. gattii but showed only a 30% reduction in one isolate of C. neoformans var. neoformans. Based on these results, the differential effect of EDTA on the two varieties of C. neoformans appeared to be due to greater inhibition of urease synthesis in C. neoformans var. gattii.

The total DNA per cell and DNA synthetic cycle phases were determined by flow cytometry in five Candida isolates including three species: Candida albicans 208R1, Candida tropicalis ATCC 750, and Candida parapsilosis 970, 3138, and ATCC 22019. The cells were prepared for flow cytometry by fixation in Carnoy fixative followed by staining with mithramycin. Marked but stable and reproducible inter- and intraspecific differences in total DNA per cell of stationary-phase cultures were found which did not correlate directly to diphenylamine estimates of the same parameter. This discrepancy was resolved by mathematically converting flow cytometry data into diphenylamine data. The reason for the discrepancy was found in studies of the DNA synthetic cycle of these yeasts: a large but isolate-specific variable proportion of the population is arrested in the S and G2-M phases after the culture passes from exponential to stationary phase. Histograms of exponential-growth-phase Candida isolates demonstrate that the majority of the population is in the G2-M phase of the DNA synthetic cycle. The DNA content of the C. tropicalis and C. parapsilosis isolates studied is as high as or higher than that of C. albicans. Extranuclear fluorescent particles were observed in the C. tropicalis isolate. No equivalent particles could be detected in the other four Candida isolates. The nature of the particles is unknown.

The taxonomy of Filobasidiella neoformans Kwon-Chung and F. bacillispora Kwon-Chung and their anamorphs were reinvestigated. Although the cross between the type cultures of the two species failed to produce viable basidio-spores, another pair of isolates did yield viable basidiospores. The segregation of phenotypic markers among the tetrads isolated from this interspecific cross proved that meiosis had occurred. On the basis of other previously known differences and the present genetic study, the two species are now considered to be two varieties of the species, F. neoformans. The anamorph of F. neoformans var. neoformans grew well at 37°C in vitro and produced fatal infection in mice while that of F. neoformans var. bacillispora grew poorly at 37°C and failed to produce fatal infection in mice. Cryptococcus bacillisporus Kwon-Chung et Bennett is regarded as a synonym of C. neoformans var. gattii Vanbreuseghem et Takashio.

Air sampling of the rooms and corridors of the oncology wards of the hospital was carried out over a 54-week period to assess the concentration of viable Aspergillus conidia. A. fumigatus and A. flavus were recovered at a mean of 1·83 cfu m-3 air sampled. Individual samplings yielded concentrations of up to 11·6 cfu m-3. Other Aspergillus spp. were recovered at a mean of 2·38 cfu m-3 (maximum 32·6 cfu m-3). Concentration was not correlated with season or hospital ward. Review of autopsy results showed an average of 6·6 cases of aspergillosis annually over a 22-year period. No seasonal variation in case incidence was found. Six cases of invasive aspergillosis were diagnosed on the three cancer wards during the air-sampling period, but no association was seen linking these cases with changes in recovery of airborne Aspergillus. A seasonal pattern was not observed in the overall incidence of aspergillosis cases nor concentrations of airborne conidia.

A polysaccharide capsule is one of the most important virulence factors for the pathogenic fungus Cryptococcus neoformans. We previously characterized two capsule-associated genes, CAP59 and CAP64. To further dissect the molecular mechanism of capsule synthesis, 16 acapsular mutants induced by 4-nitroquinoline-1-oxide were obtained. The acapsular phenotype of one of these mutants was complemented. The cloned gene was designated CAP60, and deletion of this newly described capsule-associated gene resulted in an acapsular phenotype. The proposed 67-kDa Cap60p contains 592 amino acids and appears to have a putative transmembrane domain close to the N terminus. DNA sequence analysis revealed that CAP60 has similarity to CAP59 at the center portion of its coding regions. Contour-clamped homogeneous electric field blot analysis suggested that these two genes are on the same chromosome. CAP60 and CAP59, however, could not be functionally substituted for each other by direct complementation or by domain swap experiments. In addition, CAP60 is closely linked to a gene which is similar to a cellulose growth-specific gene of Agaricus bisporus, CEL1. Immunogold electron microscopy studies of the epitope-tagged CAP60 gene revealed that Cap60p was primarily localized to the nuclear membrane. Animal model studies indicated that CAP60 is essential for virulence. Thus, CAP60 is required for both capsule formation and virulence.

The extracellular polysaccharide capsule of Cryptococcus neoformans is a well-recognized virulence factor. Strain 602 is an acapsular clinical isolate of unknown serotype which has been widely used in studies of virulence and host-parasite interactions. In previous studies, strain 602 was compared with genetically unrelated strains of various serotypes because the wild-type equivalent of strain 602 was not available. We created an encapsulated strain, TYCC38-602, by transforming strain 602 with the CAP64 gene which was isolated from a serotype D strain. Serological tests and chemical analysis of the major polysaccharide capsule of TYCC38-602 indicated that strain 602 was originally derived from a serotype A strain. Restoration of the ability to produce a capsule enabled strain 602 to cause fatal infection in mice, whereas the acapsular strain 602 remained avirulent. Capsule-restored yeast cells of strain 602 activated the human complement system and bound C3 fragments in a manner that is characteristic of encapsulated cryptococci. In addition, the capsule in TYCC38-602 masked the ability of the organism to induce tumor necrosis factor alpha and subsequent nitric oxide synthase production in primed macrophage-like cells. These results indicate that the lack of capsule in strain 602 is the reason for its inability to cause fatal infection. Moreover, the acapsular phenotype accounts for differences in various biological activities of strain 602 compared to encapsulated strains. The results also indicate that the gene product of CAP64 does not contribute to serotype specificity of capsules in C. neoformans.

The SNF1 gene of Saccharomyces cerevisiae (ScSNF1) is essential for the derepression of catabolic repression. We report here the isolation and characterization of an SNF1 homolog from Candida albicans (CaSNF1) which is apparently essential for the viability of this organism. The putative amino acid sequence of CaSNF1 has 68% identity with that of ScSNF1 and can restore the S. cerevisiae snf1 delta mutant's ability to utilize sucrose. Disruption of one of the CaSNF1 alleles resulted in morphological changes and decreased growth rates but did not modify the carbon source utilization pattern. Repetitive unsuccessful attempts to generate a snf1/snf1 homozygote by disruption of the second allele, using various vectors and approaches, suggest the lethal nature of this mutation. Integration into the second allele was possible only when a full-length functional SNF1 sequence was reassembled, further supporting this hypothesis and indicating that the indispensability of Snf1p prevented the isolation of snf1/snf1 mutants. The mutant bearing two disrupted SNF1 alleles and the SNF1 functional sequence maintained its ability to utilize sucrose and produced stellate colonies with extensive hyphal growth on agar media. It was demonstrated that in a mouse model, the virulences of this mutant and the wild-type strain are similar, suggesting that hyphal growth in vitro is not an indicator for higher virulence.

The SNF1 gene product, a serine/threonine protein kinase, is a global regulatory protein which has been isolated from several organisms. In Saccharomyces cerevisiae the SNF1 gene product is essential for the derepression of glucose repression since snf1 strains are unable to utilize sucrose, galactose, maltose, melibiose, or nonfermentable carbohydrates. Moreover, the SNF1 gene product was suggested to interact with additional regulatory pathways and to affect the expression of multiple target genes as reflected by the pleiotropic nature of the snf1 mutation. Here we report the characterization of the SNF1 homolog of Candida glabrata, a pathogenic yeast phylogenetically related to S. cerevisiae. The carbon utilization spectrum of C. glabrata is considerably narrower than that of other pathogenic yeasts, and the majority of the strains utilize solely glucose and trehalose from among 20 of the most commonly tested carbohydrates. Disruption of the C. glabrata SNF1 homolog resulted in the loss of the ability to utilize trehalose, indicating that even in an organism with such a limited carbon utilization spectrum, the regulatory mechanism governing catabolic repression is preserved.

Survey revealed that the mating type alpha is predominant among natural and clinical isolates of Cryptococcus neoformans regardless of the serotype. The ratio of alpha and a type was about 40:1 among 105 natural isolates and 30:1 in 233 clinical isolates.

The Masson-Fontana silver stain for melanin was employed for the differentiation of pathogenic fungal species in human or mouse tissues. The fungi studied were Candida albicans, Candida tropicalis, Candida glabrata (Torulopsis glabrata), Cryptococcus neoformans, Cryptococcus bacillisporus, Coccidioides immitis, Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Rhizopus rhizopodiformis, and Aspergillus fumigatus. The tissue sections stained with Masson-Fontana silver stain showed a dark brown to black color in the wall of cryptococci, whereas the walls of remaining fungal species were hyaline, except for those of S. schenckii. The yeastlike cells of S. schenckii showed faint brown pigment on the wall. Cultures of these fungi showed staining characteristics identical to those of the in vivo results. Cultures of four nonpathogenic Cryptococcus species, Cryptococcus uniguttulatus, Cryptococcus laurentii, Cryptococcus terreus, and Cryptococcus luteolus, were also tested for staining by the Masson-Fontana procedure. Of these, only C. laurentii stained positively, and the pigment on the cell wall was as intense as that of the cells of C. neoformans. These results indicate that the Masson-Fontana silver stain can be used as a specific stain in the histological diagnosis of cryptococcosis.