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PubMed2001

First report of Cryptococcus neoformans var. gattii serotype B from Greece.

A plethora of cases of Cryptococcus neoformans infections have been recorded worldwide in immunocompromised individuals. The number of such cases showed a steady rise before 1981 and increased dramatically thereafter due to the AIDS epidemic. A similar pattern has been seen in Greece but, so far, infections appear to have been caused by C. neoformans var. neoformans. This paper describes for the first time two culture-proven C. n. var. gattii serotype B infections in Greece, one in an AIDS patient and one in a patient with systemic lupus erythematosus.

Acquired Immunodeficiency Syndrome↗
PubMed2001

An immunohistochemical method that differentiates Cryptococcus neoformans varieties and serotypes in formalin-fixed paraffin-embedded tissues.

An immunohistochemical method for determining the variety of Cryptococcus neoformans in formalin-fixed paraffin-embedded tissues was developed using mAbs 471, 302 and CRND8. The method was validated primarily using veterinary patients for which both formalin-fixed lesions and a cultured isolate were available. L-Canavanine glycine bromothymol blue (CGB) agar and the 'Crypto-Check' kit were used to determine the variety and serotype, respectively, of cultured isolates. Immunohistochemistry accurately predicted the C. neoformans variety in all tissue specimens. The CGB agar method of determining C. neoformans variety gave the same result as immunohistochemistry for 30/31 specimens. For the single discordant isolate, the serotype, random amplification of polymorphic DNA profile, microscopic and colony morphology all supported the immunohistochemical staining pattern in suggesting C. neoformans var. gattii; however, the CGB agar result was at variance. Of the C. neoformans var. neoformans cases, immunohistochemistry was congruent with variety for 13/13 cases and with serotyping for 10/13 cases. The three discordant cases were classified as having some serotype D reactivity by immunohistochemistry, but were considered to be serotype A using the Crypto-Check kit. This new method should prove a valuable epidemiological tool in studies of cryptococcosis, especially in the veterinary setting where archival tissue specimens may exist but corresponding mycological data is typically absent. The versatility of this method will expand in the future as other monoclonal antibodies with different specificities are developed.

Antibodies, Monoclonal↗
PubMed2002

An auxotrophic pigmented Cryptococcus neoformans strain causing infection of the bone marrow.

Cryptococcosis, caused by an encapsulated fungus, Cryptococcus neoformans, has emerged as a life-threatening infection in HIV-positive individuals and other immunocompromised hosts. This report describes an unusual strain of C. neoformans isolated from an AIDS patient that developed pigment on Sabouraud's medium. The yeast was auxotrophic for adenine due to a deletion in the coding region of ADE2, and was complemented by introduction of a functional copy of the ADE2 gene from C. neoformans. The yeast had an unusual myelotropism that was clinically evident as a pancytopenia with displacement of bone marrow precursors by yeast cells, and it had an unusual spectrum of infection in the human host. This is the first description of a nutritional auxotroph of C. neoformans isolated from a patient.

AIDS-Related Opportunistic Infections↗
PubMed2002

Effect of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on polymorphonuclear neutrophils, monocytes or monocyte-derived macrophages combined with voriconazole against Cryptococcus neoformans.

The antifungal activity of voriconazole (VCZ) was tested against Cryptococcus neoformans (Cn) with and without the addition of polymorphonuclear neutrophils (PMN), monocytes or monocyte-derived macrophages (MDM) in vitro. Human effector cells with and without the addition of VCZ were incubated with Cn for 24 h. PMN, mono and MDM alone resulted in 61%, 34% and 23% inhibition of Cn, respectively (n = 3, P<0.01). VCZ at 0.01 and 0.05 microg ml(-1) alone resulted in 48% inhibition and 19% killing (n = 6). The addition of VCZ at 0.01 and 0.05 microg ml(-1) to human effector cells enhanced killing of Cn by 51% and 71% for the PMN, 41% and 58% for the mono, and 14% and 34% for the MDM, respectively. The addition of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) significantly enhanced the ability of human effector cells to kill Cn. G-CSF and GM-CSF plus PMN resulted in 47% and 46% killing, respectively; GM-CSF plus monocytes or MDM resulted in 31% or 22% killing, respectively. G-CSF and GM-CSF further enhanced the collaborative killing effect of human effector cells and VCZ. At 0.01 and 0.05 microg ml(-1) of VCZ, G-CSF or GM-CSF enhanced PMN killing to 92% and 93% or 87% and 94%, respectively. GM-CSF enhanced both mono and MDM with VCZ at 0.01 and 0.05 microg ml(-1) in killing Cn to 62% and 86%, and 61% and 84%, respectively. These results suggest that VCZ would have good efficacy in the treatment of Cn infection in humans. Furthermore, VCZ would have enhanced efficacy in clinical settings where either G-CSF or GM-CSF was being used.

Antifungal Agents↗
PubMed2002

Environmental sampling for Cryptococcus neoformans var. gattii from the Blue Mountains National Park, Sydney, Australia.

The environmental association of Cryptococcus neoformans var. gattii with decaying wood in tropical and subtropical regions of the world is well documented. In Australia, the yeast appears confined to certain species of Eucalyptus or very closely related tree species. In this study, we attempted to isolate C. n. var. gattii from different gum tree species in the Blue Mountains National Park. Out of 99 samples from 9 different tree species, only 3 yielded viable yeast colonies; 2 were from turpentine gums (Syncarpia glomulifera) and 1 was from a decayed stump of an unknown species. All of the colonized trees occurred in close proximity in urbanized areas of the Park, and all isolates shared identical DNA fingerprinting profiles. We suggest that domestic animal vectors may be responsible for the introduction and transmission of the yeast in this region, but that propagation and dispersal are very limited. This study indicates that C. n. var. gattii may occur on trees and in areas that were not previously expected to host it. However, the low incidence in the Blue Mountains National Park means this yeast is unlikely to pose any hazards to humans and animals living in or visiting this area.

Cryptococcus neoformans↗
PubMed2002

Cryptococcus neoformans in the koala (Phascolarctos cinereus): colonization by C n. var. gattii and investigation of environmental sources.

This study is the one in a series looking at the relationship among Cryptococcus neoformans var. gattii, koalas and the environment. The koala was used as a natural biological sampler in an attempt to understand the dynamics of C. neoformans var. gattii in Australian environments. Evidence of asymptomatic nasal and skin colonization for extended periods by large numbers of C. n. var. gattii was obtained and geographical factors assessed. The key finding was the ability of koalas to amplify numbers of C. n. var. gattii in certain environments. Koalas were not found to be obligatory for the survival of the organism in all environments. Geographical factors alone could not explain differing rates of nasal and skin colonization in koalas in different environments. A strong association between healthy koalas and C. n. var. gattii was confirmed and C n. var. gattii was isolated from novel sources, including the turpentine gum tree (Syncarpia glomulifera), tallowwood (Eucalyptus microcorys) and flooded gum (E. grandis). It seems likely that as yet undiscovered environmental sources of C. n. var. gattii exist in eastern Australia. Further investigation of host, environmental and organism factors integral to the hostpathogen relationship will assist an understanding of the progression from colonization to tissue invasion and cryptococcosis in all species.

Animals↗
PubMed2002

Cryptococcus neoformans var. gattii in the koala (Phascolarctos cinereus): serological evidence for subclinical cryptococcosis.

Cryptococcus neoformans var. gattii has been shown to have a strong association with eucalypts frequently used by koalas and, not surprisingly, it has been shown to colonize the nasal cavities of koalas. The progression from nasal colonization to tissue invasion is critical to understanding the pathogenesis of cryptococcosis in this species and provides a model for pathogenesis of cryptococcosis in other species. Cryptococcal antigenaemia was detected in twenty-eight healthy koalas from three different regions. This was interpreted as representing limited subclinical disease. One koala developed cryptococcal pneumonia 6 months after leaving the study, whereas another developed cryptococcal meningoencephalitis during the course of the study. Opportunistic necropsies on ten antigen-positive koalas resulted in discovery of small cryptococcal lesions in two (paranasal sinus and lung, respectively). Our data suggest that cryptococcal antigenaemia occurs commonly in koalas, especially in areas with a high environmental presence of C n. var. gattii. Subclinical disease appears most likely to manifest as a small focal lesion in the respiratory tract. Possible outcomes include elimination by an effective immune response, quiescence with possibility of later re-activation or direct progression to overt disease. Symptomatic and subclinical cases showed differences in levels of antigenaemia. The data presented have significant implications for koalas in captivity.

Agglutination Tests↗
PubMed2002

Cryptococcus neoformans pulmonary granuloma formation is associated with matrix metalloproteinase-2 expression.

The aim of this study was to investigate matrix metalloproteinase (MMP) expression during the immune response to pulmonary Cryptococcus neoformans (Cne) infection. The immune response generated in C.B-17 and C57BL/6 mice to pulmonary Cne infection has previously been characterized as type 1 and type 2, respectively, differing in the cytokines produced and leukocytes recruited during infection, influencing the extent of Cne clearance from the lung. The focus of this study was to examine changes in expression of MMP-2 and tissue inhibitor of metalloproteinase (TIMP)-2 in the lungs of Cne-infected mice during the two types (type 1 vs. type 2) of responses. C.B-17 mice that formed well-defined granulomas had elevated levels of pulmonary MMP-2 mRNA early during infection. C57BL/6 mice that had poorly defined cellular aggregates did not express detectable levels of pulmonary MMP-2 mRNA until later in the infection. Specific expression of MMP/TIMP was correlated with the type of immune response present, resolution of Cne infection and the resulting lung pathology.

Animals↗
PubMed2002

Determination of the pH of the Cryptococcus neoformans vacuole.

We have previously demonstrated the antifungal activity of the weak bases chloroquine and quinacrine against Cryptococcus neoformans. Quinacrine, being fluorescent, was seen to be concentrated within a complex vacuolar structure within the cryptococcal cell. Here we determined the pH of this compartment using the pH-sensitive fluorescent dye, 5-(and 6-) carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA). Carboxy-DCFDA was concentrated within the cryptococcal vacuole, giving a pattern of fluorescence similar to that previously observed with quinacrine. For each experiment, a standard curve of fluorescence ratio against pH was generated using buffers of defined pH containing a mixture of ionophores and inhibitors to equilibrate vacuolar pH with that of the medium. The pH of the cryptococcal vacuole of five strains was calculated to range from 5.3 to 5.9 with a mean of 5.6. This acidic pH is consistent with a model in which weak bases such as chloroquine and quinacrine are accumulated, by ion trapping within the fungal vacuole. Antifungal activity may result from the consequent disruption of pH-dependent processes as well as effects on other as yet undefined fungal targets.

Cryptococcus neoformans↗
PubMed2002

A flucytosine-resistant Cryptococcus neoformans (serotype D) strain isolated in turkey from cutaneous lesions.

A Cryptococcus neoformans strain from cutaneous lesions of a patient with thrombotic thrombocytopenia purpura was tested for in vitro susceptibility against seven conventional antifungal agents. The strain was susceptible to fluconazole, itraconazole, ketoconazole and miconazole but was resistant to 5-fluorocytosine (5-FC). Minimal inhibitory concentration (MIC) values obtained against amphotericin B and terbinafine were 1 and 4 microg ml(-1), respectively. The isolate belonged to serotype D. Few human cases of cryptococcosis have been reported over the last 50 years in Turkey. This is the first C. neoformans isolate in Turkey shown to have primary resistance to 5-FC. Primary resistance to flucytosine is rarely reported in C. neoformans and may be associated with treatment failure in some cases.

Adult↗
PubMed2002

Epidemiological aspects of clinical and environmental Cryptococcus neoformans isolates in the Brazilian state Rio Grande do Sul.

Cryptococcus neoformans is a pathogenic fungus that causes life-threatening meningoencephalitis in immunocompromised patients (HIV-positive patients), and lymphoproliferative disorders in patients subjected to organ transplantation and other immunosuppressive therapies. This fungus is commonly found in soil and avian excreta, mainly from pigeon and turkey. We describe the isolation and characterization of 17 clinical and 10 environmental (pigeon excreta) isolates from the Brazilian state Rio Grande do Sul. We analyzed capsule formation, carbon assimilation pattern, canavanine-glycine-bromothymol blue (CGB) reaction, and nitrate and urease tests, as well as susceptibility to antifungal drugs. The genetic variability among C. neoformans isolates was studied using randomly amplified polymorphic DNA (RAPD) analysis. Eight of 22 arbitrary polymerase chain reaction primers used confirmed genetic polymorphism among the environmental isolates tested, suggesting that it remains feasible to use RAPD analysis as a typing method. Three of the selected primers yielded 10 molecular subclasses. The majority of the clinical isolates were assigned to the molecular subclass F. The RAPD data obtained reinforce the developing consensus about the population structure of this fungus.

AIDS-Related Opportunistic Infections↗
PubMed2002

Role of mitochondrial carrier protein Mrs3/4 in iron acquisition and oxidative stress resistance of Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic basidiomycete that causes meningitis in immunocompromised patients. In this paper, we demonstrate that a previously described oxidant-sensitive mutant, oxy1, has constitutive ferric reductase and iron uptake, similar to a ferric reductase regulatory mutant, frr1. Through meiotic genetic analysis, we show that the two mutations are allelic. By complementation of frr1 with a genomic library, we isolated a gene, MRS3/4. The encoded protein is a putative solute transporter of the inner mitochondrial membrane. Disruption of this gene led to high ferric reductase, iron uptake and iron content, as well as increased sensitivity to hydrogen peroxide and slow growth in low iron medium. The disrupted gene is allelic with oxy1 and frr1. We sequenced the oxy1 and frr1 alleles of MRS3/4 and found that the frr1 mutation results in a premature stop codon, while the oxy1 mutation results in the substitution of a highly conserved glutamate residue with lysine. The Mrs3/4 protein appears to be involved in mitochondrial iron transport in eukaryotes. Resistance to strong oxidants requires stringent control of iron metabolism.

Amino Acid Sequence↗
PubMed2003

Pathogenicity of Cryptococcus neoformans var. gattii in an immunocompetent mouse model.

The pathogenicity of two different genomic profiles of Cryptococcus neoformans var. gattii serotype B isolated from goats that died from cryptococcal pneumonia was assessed in an experimental model of immunocompetent mice. One strain of each randomly amplified polymorphic DNA (RAPD) profile (GR52 and GR56) and three reference C. neoformans isolates representing serotypes B, D and C were used. BALB/c male mice were inoculated by the intraperitoneal route with each strain. After 4 weeks of follow-up, the animals were sacrificed and autopsy specimens of testes, liver, spleen, kidney, lungs and brain were cultured and stained for histopathology. Although spontaneous mortality was only 2% (one animal), all mice except for those inoculated with serotype C showed positive cultures in almost one organ. The strain GR52 isolated from goat showed the highest rate of positive cultures (80%) followed by serotype D (77%). Serotype B reference strain and second goat strain GR56 were both isolated from 70% of samples. Serotype C was recovered in only 33% of organs, and never from brain or lung specimens. GR52 grew abundantly from all lung cultures, and yeast cells with large capsules were seen in histopathology inside the alveoli, peribronchial vessels and interalveolar spaces. They appeared to elicit no inflammatory response. We conclude that intraperitoneally inoculated C. neoformans var. gattii shows high virulence in this immunocompetent mouse model. Strain GR52 was highest in pathogenicity and had marked lung tropism. In contrast, the serotype C reference strain showed the lowest pathogenicity and seemed not to spread outside the abdominal viscera.

Animals↗
PubMed2003

Isolation of two molecular types of Cryptococcus neoformans var. gattii from insect frass.

Cryptococcus neoformans var. gattii has regularly been the cause of serious human disease. However, the environmental sources of these infections often remain unclear. During an environmental sampling study, two different strains of C. neoformans var. gattii were isolated from fresh insect frass (order Lepidoptera; family Oecophoridae) in a shallow cavity in the bark of a living Eucalyptus tereticornis tree, one molecular type VGI and the other VGII. This is the first published report of the isolation of two different molecular types of C. neoformans var. gattii from a single source, and the third of isolation of molecular type VGII from an environmental source. The potential association with insect frass is consistent with categorising C. neoformans var. gattii within the Tremellales, containing mycoparasitic fungi.

Animals↗
PubMed2002

Treatment of experimental nephrosis by culture filtrate of Cryptococcus neoformans var. gattii (CneF).

The therapeutic effect of the culture filtrate of cryptococcus neoformans var. gattii (CneF) was tested in Adriamycin-induced nephropathy. The CneF was administered at different doses (36, 54 and 90 mg/kg based on carbohydrate concentration), one i.p. injection every 72 hours for a total of 10 injections. The treated patient rats showed a significant reduction in proteinuria, plasma cholesterol concentration, BUN and significant increase in urine creatinine levels. Moreover, treatment with CneF significantly reduced number of glomerular leukocytes and decreased the tubular casts. These data suggest that CneF therapy can ameliorate proteinuria, hypercholesterolemia and suppress the progression of glomerular lesions in experimental model of nephrosis.

Animals↗
PubMed1991

Intrapulmonary growth and dissemination of an avirulent strain of Cryptococcus neoformans in mice depleted of CD4+ or CD8+ T cells.

The contribution of T lymphocyte subpopulations to intrapulmonary and systemic resistance against an opportunistic strain of Cryptococcus neoformans was examined. It was found that C. neoformans was destroyed when introduced into the lungs of normal mice, but disseminated to the brains of mice treated with an antibody that depleted them of CD4+ T cells. Depletion of either CD8+ or CD4+ T cells impaired the ability of the host to clear the yeast from the lung. These results, together with the observation that CD8+ T cells accumulate in the lungs of CD4+ T cell-deficient mice, suggest that CD8+ T cells play an important role in resistance to C. neoformans infection acquired via the respiratory tract.

Animals↗