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Case Reports |
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Abstract |
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Key Words: Cryptococcus • dogs • fungal infection • genetic analysis
Cryptococcus gattii (formerly Cryptococcus neoformans var. gattii) is a haploid yeast formerly thought to be restricted to tropical and subtropical climates.1,9–11 Infection is by inhalation of airborne propagules.10,11 The pathogenicity of C. gattii differs from that of Cryptococcus neoformans in that C. gattii infection occurs most often in immunocompetent hosts.1,10,11 Genotypic analysis of C. gattii has revealed 4 major molecular groups: VGI–VGIV.6 Cryptococcus gattii has recently emerged as a human and animal pathogen, causing an outbreak on Vancouver Island, Canada, that began in 1999 and was ongoing at the time of this writing.1–3,12,13 Molecular studies indicate a previously rare genotype to be the primary cause of the outbreak.6,9 Molecular analysis of the mating type–specific genes SXI1
and SXI1a of this genotype suggest that same sex-mating has resulted in a highly pathogenic strain within this region.6 More recently, C. gattii infection has caused clinical disease in animals and humans elsewhere in the Pacific Northwest.3,4,10 A confirmed case of C. gattii caused by the Vancouver Island outbreak major genotype, designated VGIIa, was found in a man from Puget Sound Washington, USA, in January 2006.13 Cryptococcus gattii infection with the VGIIa genotype has also been reported in cats in Washington State, with no apparent link to Vancouver Island.10
Cryptococcus gattii and C. neoformans grow readily on Sabouraud dextrose agar. Colonies can be smooth, mucoid, or wrinkled, with increased virulence associated with a mucoid or wrinkled phenotype.8 Colony phenotype is related to the composition of the polysaccharide capsule, and phenotypic switching has been documented in laboratory studies of C. neoformans7 and C. gattii.8 Phenotypic alteration of C. gattii also occurs in vivo, and alteration in the polysaccharide capsule is related to the ability to cross the blood–brain barrier during infection of the central nervous system.8 This report describes a case of primarily central nervous system infection by C. gattii with bimorphic colony types in a dog from Western Oregon with no history of travel to Canada. Both isolates were confirmed to be the Vancouver Island outbreak genotype.
A 1.5-year-old, intact, female Keeshond dog was presented for necropsy. The dog was born in Hillsboro, Oregon, and had been relocated to Salem, Oregon, at the age of 7 weeks. The dog had traveled to Washington State, including visits to Seattle and the Puget Sound area of Washington, but there was no travel history to Vancouver Island or Canada. The dog died after an approximately 1-week history of cervical pain, ataxia, anorexia, vomiting, depression, and collapse. Gait abnormalities were characterized by bending and staggering to the left. Additional history indicated occasional bouts of lethargy, diarrhea, and vomiting for approximately 3 months before the recent severe illness. Vaccinations and deworming were current.
The dog weighed 11.4 kg and was in poor body condition. The most significant finding was a yellow, soft, gelatinous mass (approximately 3 cm x 2.5 cm x 1.5 cm) within the left cranial vault, loosely attached to the underside of the left cerebrum at the area of the left olfactory bulb. A cavitation within the adjacent brain exuded clear yellow viscous fluid when sectioned. The mass was adhered out to the inner surface of the cranium and extended to the cribriform plate, but did not involve the nasal cavity. Cytologic evaluation revealed heavily encapsulated yeast with narrow-based budding consistent with Cryptococcus spp. A swab of the left caudal nasal cavity and a swab of the mass in the cranial vault were obtained and pooled for fungal isolation. The lungs contained patchy red areas and mesenteric lymph nodes were diffusely enlarged (approximately 2–3 times normal). No other postmortem findings were significant. The spinal cord was not examined. The brain and attached mass and tissue samples from lung, kidney, pancreas, liver, adrenal gland, spleen, thyroid gland, intestine, and mesenteric lymph node were fixed in 10% neutral buffered formalin and prepared for routine histopathologic examination. Selected sections of the granulomatous intracranial mass, kidney, and lung were also stained with Mayer's mucicarmine, Grocott's methenamine silver (GMS), and periodic acid–Schiff (PAS) stains.
Sections of the left cranial mass and cerebrum revealed severe granulomatous meningitis and encephalitis with numerous encapsulated spherical fungal organisms approximately 4–20 µm in diameter exhibiting narrow-based budding (Figs. 1, 2). Collagen was prominent within the granulomatous mass external to the brain, indicative of chronicity. Small multifocal to locally extensive zones of granulomatous inflammation with similar fungal organisms were detected in the renal cortices, with a very small number of similar foci within the pancreas and lung. Mesenteric lymph nodes were hyperplastic with no evidence of fungal infection, and fungal infection was not detected in any other organ section examined. Fungal organisms stained with PAS and GMS stains, and the capsule stained intensely with Mayer's mucicarmine stain (Fig. 2). No variation in fungal morphology was detected in tissue sections.
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In addition to phenotypic analysis, multilocus sequence typing at 8 unlinked loci previously shown to characterize C. gattii outbreak strains was applied to the variant colony morphologies as previously described.6 Both isolates harbored the SXI1 gene, and were therefore of the mating type.6 Furthermore, both isolates were identical across all 8 gene loci tested and were of the VGIIa molecular type, the major Vancouver Island genotype (Fig. 5).
The clinical signs in this case were primarily neurologic and were consistent with the pathologic findings. Central nervous system infection is common in dogs with C. gattii infection, and the presence of clinical signs of central nervous system disease has been shown to be a significant predictor of mortality.3 No underlying disease process that might decrease immune function was identified in this dog, consistent with prior reports of C. gattii infection in immunocompetent hosts.1,10,11
It is suggested that the bimorphic colony types might reflect differences in the nature of the organism in the intracranial granuloma versus that present in the nasal cavity. Perhaps 1 isolate infected the animal, and then an in vivo colony phenotypic switch either resulted in dissemination or occurred as a consequence of dissemination. The swabs from both areas were pooled for culture, however, and this remains a hypothesis. Whether phenotypic switching is genetically or epigenetically controlled is not yet known. This case illustrates that phenotypic variants isolated from a single patient can differ in melanin production and in mating ability while maintaining similar genetic profiles.
The majority of the infection in this case was adjacent to the cribriform plate in the cranium and in the left cranioventral cerebrum. Subclinical nasal infection by C. gattii in dogs and cats can progress to systemic infection,4 which is the suspected pathogenesis in this case. This case illustrates the recent emergence of C. gattii in both veterinary and human patients in the Pacific Northwest. The genetic concordance of this isolate with the outbreak major genotype indicates that this case is an expansion of the geographic range of this virulent strain from Vancouver Island to the Pacific Northwest of the United States. An alternative hypothesis is that the Vancouver Island VGIIa major genotype was present in both Vancouver Island and the United States and emerged independently in the 2 locales. However, because no cases in animals or patients in Washington and Oregon were reported from 1999 until the first index case in 2006 in the San Juan Islands, during which time hundreds of cases occurred on Vancouver Island in animals and humans,10 the more parsimonious explanation is favored that this represents an expansion from Vancouver Island, Canada, into the United States. Moreover, ample evidence indicates that the organism is spread via a variety of routes, including human traffic,1 and the spate of several dozen recent cases in animals and humans in Washington and Oregon during 2007–2008 (Byrnes, unpublished observations) is in accord with models positing a recent expansion of geographic range by introduction from Vancouver Island, Canada, into the United States. This case also reflects an important need for genotypic analysis of veterinary isolates of Cryptococcus spp. infections to determine both the species causing infection and the molecular type of the isolate to establish modes and patterns of this emerging pathogenic fungus in the Pacific Northwest.
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a Hardy Diagnostics, Santa Maria, CA.
c British Columbia Cryptococcal Working Group (http://www.cher.ubc.ca/cryptococcus/new/methods.htm).
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This article has been cited by other articles:
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  R. Velagapudi, Y.-P. Hsueh, S. Geunes-Boyer, J. R. Wright, and J. Heitman Spores as Infectious Propagules of Cryptococcus neoformans Infect. Immun., October 1, 2009; 77(10): 4345 - 4355. [Abstract] [Full Text] [PDF]
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 H. Ma, F. Hagen, D. J. Stekel, S. A. Johnston, E. Sionov, R. Falk, I. Polacheck, T. Boekhout, and R. C. May The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation PNAS, August 4, 2009; 106(31): 12980 - 12985. [Abstract] [Full Text] [PDF]
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