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School of Molecular & Biomedical Science
The University of Adelaide
AUSTRALIA 5005

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Dr David Ellis
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Cryptococcus gattii

Teleomorph: Filobasidiella bacillispora

Cryptococcus gattii has two serotypes (B and C) and has recently been reclassified as a separate species (Kwon-Chung et al. 2002). C. gattii generally has a more restricted geographical distribution than C. neoformans, causing human disease in climates ranging from temperate to tropical Australia, Papua New Guinea, parts of Africa, India, southeast Asia, Mexico, Brazil, Paraguay and Southern California, although recent infections have also been reported from Vancouver Island, Canada.   Non-immunocompromised hosts are usually affected and large mass lesions in the lung and/or brain (cryptococcomas) are often present (Sorrell, 2001).

Canavanine-glycine-bromthymol blue (CGB) agar is now the medium of choice to determine the species status between C. gattii and C. neoformans isolates. This simple biotype test is based on the ability of C. gattii isolates to grow in the presence of L-canavanine and to assimilate glycine as a sole carbon source.

blue and yellow results on agar plates
CGB agar turns blue for Cryptococcus gattii.

brown and white colonies on agar plate
Bird seed agar plate showing brown colonies of C. gattii and white colonies of Candida albican

capsules of C. gattii
India ink preparation showing capsules of C. gattii.

Culture: Colonies are cream colored, smooth, mucoid yeast-like in appearance.

Microscopic morphology: Globose to ovoid budding yeast-like cells or blastoconidia, 3.0-7.0 x 3.3-7.9 um in size.

India Ink Preparation: Positive - distinct, wide gelatinous capsules surrounding the yeast cells are present.

Dalmau Plate Culture on Cornmeal and Tween 80 Agar: Budding yeast cells only. No pseudohyphae present.

Bird seed agar or caffeic acid agar: Colonies of both varieties turn dark brown in color; C. neoformans selectively absorbs a brown pigment from these media.

Canavanine-glycine-bromthymol blue (CGB) agar: turns blue within 2-5 days (use a heavy inoculum).

Physiological Tests:

Germ Tube test is Negative
Hydrolysis of Urea is Positive
Growth on Cycloheximide medium is Negative
Growth at 37C is Weakly Positive

Fermentation Reactions: Where fermentation means the production of gas and is independent of pH changes.

Negative: Glucose; Sucrose; Lactose; Galactose; Maltose; Trehalose.

Assimilation Tests:

Positive: Glucose; Galactose; Maltose; Sucrose; Trehalose; D-Xylose; Melezitose; Raffinose (weak); Cellobiose (weak); Inositol; L-Rhamnose; D-Arabinose; L-Arabinose (delayed); D-Ribose (weak); Galactitol (delayed); D-Mannitol; D-Glucitol.
Variable: Soluble Starch; Glycerol; Succinic acid; Ribitol; L-Sorbose; Salicin; Citric acid; DL-Lactic acid.
Negative: Potassium nitrate; Lactose; Melibiose; Erythritol.

 

MIC data is limited.  Antifungal susceptibility testing of individual strains is recommended.

Antifungal MIC ug/mL Antifungal
MIC ug/mL
Range
MIC90
Range
MIC90
Fluconazole
1->64
16
Amphotericin B
0.03-64
0.25
Itraconazole
0.03-1
0.25
Flucytosine
0.03-0.125
4
Voriconazole
0.03-2
0.25
Posaconazole
0.03-0.5
0.25

 

Key Features: encapsulated yeast; absence of pseudohyphae; growth at 37C; positive hydrolysis of urea; negative fermentation of sugars and positive assimilation of glucose, maltose, sucrose, galactose, trehalose, raffinose, inositol, cellobiose, rhamnose, arabinose, melezitose and xylose, and negative assimilation of nitrate, lactose, melibiose, erythritol and soluble starch; growth on bird seed (Guizotia abyssinica seed) or caffeic acid agar - colonies turn a dark brown colour; growth on CGB agar turning it blue within 2-5 days.   RG-2 organism, however mating experiments for the production of basidiospores should be done in an appropriate pathogen handling cabinet.

Clinical significance:

The distribution of cryptococcosis due to Cryptococcus gattii is geographically restricted, non-immunocompromised hosts are usually affected, large mass lesions in lung and/or brain (cryptococcomas) are characteristic and morbidity from neurological disease is high. Human disease is endemic in Australia, Papua New Guinea, parts of Africa, the Mediterranean region, India, south-east Asia, Mexico, Brazil, Paraguay and Southern California.

Environmental isolations, initially from the Barossa Valley in South Australia have established that C. gattii has a specific ecological association with Eucalyptus camaldulensis, a species of red gum widely distributed in mainland Australia. Subsequently, another species of red gum E. tereticornis was confirmed as a natural habitat. This species has a more restricted distribution occurring along the eastern coastal seaboard of Australia, extending to Papua New Guinea. More recently, high concentrations of C. gattii have been isolated from single specimens of three additional eucalypts, Eucalyptus rudis (flooded gum), E. gomphocephala (tuart) and E. blakelyi (Blakely's red gum).

Three of these species (E. camaldulensis, E. tereticornis and E. gomphocephala) have been exported to several countries in which human disease due to C. gattii has been reported though the association is not exact. Outside of Australia limited isolations of C. gattii have been made from E. camaldulensis trees growing in California, Apulia, Italy and in northern India.

Evidence for an epidemiological association between this cryptococcal habitat and human infection is circumstantial. There is correlation between the global distribution of human infection with C. gattii and the five species of eucalypts and environmental searches conducted in Australia and elsewhere have so far failed to identified any other natural source.

In Australia, 92% of human isolates and all of those from koalas (a native animal which live in close association with eucalypts) and from all five host eucalypts identified to date (ie E. camaldulensis, E. tereticornis, E. rudis and E. gomphocephala), exhibit the same genetic fingerprint (VGI) when identified by random amplification of polymorphic DNA (RAPD) and PCR fingerprinting. This finding is consistent with an epidemiological association between mammalian disease and exposure to host eucalypts. However, the occurrence of human C. gattii infection in other countries which lack the host trees, and our own observations of a distinct genetic type (VGII) in certain locations in Australia suggest that additional environmental niches for this fungus are yet to be discovered.

Eucalypt agar showing growth
Growth of C. gattii on Eucalypt agar

Mycosis: Cryptococcosis

Further reading:

Kreger-Van Rij, N.J.W. (ed) 1984. The Yeasts: a taxonomic study. 3rd Edition. Elsevier Science Publishers B.V., Amsterdam, The Netherlands.

Kwon-Chung, K.J. and J.E. Bennett. 1992. Medical Mycology. Lea & Febiger, Philadelphia and London.

Kwon-Chung K.J., Polacheck I., and Bennett J.E. 1982. Improved diagnostic medium for separation of Cryptococcus neoformans var. neoformans (Serotypes A and D) and Cryptococcus neoformans var. gattii (Serotypes B and C). J. Clin. Microbiol. 15(3):535-537.