# Cryptococcus gattii: An Emerging Cause of Fungal Disease in North America

Authors: Ashwin Dixit; Scott F. Carroll; Salman T. Qureshi
Journal: Interdisciplinary Perspectives on Infectious Diseases
 (2009)

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## Abstract

During the latter half of the twentieth century, fungal pathogens such asCryptococcus neoformans were increasingly recognized as a significant threat to the health of immune compromised populations throughout the world. Until recently, the closely related species C. gattii was considered to be a low-level endemic pathogen that was confined to tropical regions such as Australia. Since 1999, C. gattii has emerged in the Pacific Northwest region of North America and has been responsible for a large disease epidemic among generally healthy individuals. The changing epidemiology of C. gattii infection is likely to be a consequence of alterations in fungal ecology and biology and illustrates its potential to cause serious human disease. This review summarizes selected biological and clinical aspects of C. gattii that are particularly relevant to the recent North American outbreak and compares these to the Australian and South
American experience.


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## Body

## 1. Introduction

Although less than 500 of the estimated 1.5 million species of fungi
pose a threat to humans and animals [1, 2], the prevalence of fungal infections has risen over the last
century due to a progressive increase in the number of debilitated individuals.
Impaired immunity against fungi may result from one or more factors including
malignancy, advanced or severe comorbid disease, or the use of cytotoxic drugs
and broad-spectrum antibiotics. Species of Candida remain the most common cause of invasive yeast infection; however,
opportunistic filamentous fungi such as Aspergillus spp, Fusarium spp, Scedosporium spp, Penicillium spp, and the zygomycota are becoming more prevalent in oncology and transplant centers [3, 4].
Human disease resulting from environmental exposure to the basidiomycetous
yeast Cryptococcus neoformans increased
significantly since the onset of the HIV epidemic and continues to be common
among individuals that do not have access to effective antiretroviral therapy [5, 6]. C. gattii, a closely related species
that was traditionally associated with tropical and subtropical climates, is
now gaining prominence as a cause of human and veterinary disease in North
America. While most clinical cases have occurred among generally healthy
individuals that reside in the Pacific Northwest, a few tourists or visitors to
the region have also been affected. The purpose of this review is to summarize
important biological and clinical characteristics of C. gattii that are relevant to the understanding of human disease
caused by this emerging fungal pathogen.


## 2. Identification

C. neoformans was first isolated in 1894 from fermented peach juice by the Italian
Francesco Sanfelice [7]. Since that time, this organism has been recovered from numerous
locations throughout the world where its main ecological niche is soil,
particularly in association with pigeon excreta [8–10].
Cryptococci grow as unicellular, encapsulated cells in the asexual state or as
basidiomycetous filaments in the sexual state [1, 11]. Infection due to this
opportunistic fungus is believed to occur by inhalation and primarily targets
the lung with frequent dissemination to the central nervous system as well as a
variety of other organs [12, 13].C. gattii was first isolated from a leukemic patient in 1970 and described as
a variant of C. neoformans [14].
C. gattii is closely related to C.
neoformans, although its distribution is not global. C. gattii is typically restricted to tropical and subtropical
geographical regions such as Australia, Brazil, and southern California [15, 16]. In
the laboratory, these two cryptococcal species can be distinguished on the
basis of their capsular serotype: C.
gattii belongs to serotypes B and C, while C. neoformans belongs to serotypes A and D [17]. Like C. neoformans, C. gattii typically causes pneumonia and
meningitis [15]. However, C. gattii appears to have a greater propensity to infect immune competent humans [18, 19].For reasons that are not yet fully understood,C. gattii has acquired the ability to colonize new biogeoclimatic
regions and is responsible for a recent outbreak of infection among humans and
animals in the temperate climate of Vancouver Island, British Columbia (BC),
Canada [20]. Between 1999 and 2006, 171 human cases of C. gattii infection were identified, including 8 fatalities [21]. Between 2002 and 2005, the incidence of C. gattii infection on Vancouver Island peaked at 36 cases/million
people/year, a number that was significantly higher than the 0.94 cases/million
people/year observed in endemic regions of Australia [22, 23]. The
most common clinical manifestation of C.
gattii infection on Vancouver Island was pneumonia [21]. Although the majority of human infections have been found on the
east coast of Vancouver Island [23], clinical cases have also been reported on the BC mainland, Alberta,
and the states of Oregon and Washington [22, 24, 25].
Given the ongoing spread of C. gattii infection on the Pacific coast of North America, it will be important for
clinicians and laboratory scientists to remain vigilant for diseases that may
be caused by this fungal pathogen.


## 3. Taxonomy

Cryptococcus is a largely polyphyletic genus that consists of at least 37
different species and belongs to the kingdom Fungi, phylum Basidiomycota,
class Tremellomycetes, and order Tremellales [26–28]. The
subclassification of this genus has been the subject of much debate and
modification, particularly in response to the development of newer molecular
typing methods [26, 27, 29]. The C. neoformans species complex was first
classified according to structural variations of the extracellular
polysaccharide capsule that are distinguished by agglutination assays with
antigen-specific antibodies [30, 31].
Using this approach, C. neoformans was
classified into four serotypes, A through D, in the 1950s and 1960s [30, 32]. The
hybrid serotype AD is often considered to be a fifth serotype of the C. neoformans species complex, and rare
hybrid serotypes between C. neoformans and C. gattii such as BD and AB have
also been observed [33–37].C. gattii was initially classified as a variety of C. neoformans, bearing the name C.
neoformans var. gattii or var. bacillispora [15, 38].
Subsequently, C. gattii and C. neoformans were shown to have
substantial differences in their biochemistry, ecology, epidemiology, and
clinical manifestations (see review [15]) as a result of the divergence of serotypes A and D from serotypes
B and C that occurred approximately 37 million years ago [15, 39].
Following a series of revisions to the classification of the C. neoformans species complex [11, 14, 17, 38, 40, 41],
cryptococci are now divided into two major species, C. neoformans (serotypes A, D, and AD) and C. gattii (serotypes B and C). The sexual state of C. gattii is known as Filobasidiella bacillispora [38, 41].The most recent
classification of cryptococci was established by genetic typing using PCR
fingerprinting, random amplification of polymorphic DNA (RAPD), amplified
fragment length polymorphism (AFLP) analysis and multilocus sequence typing
(MLST) [17, 23, 35, 37, 42–45].
Based on genetic diversity, cryptococci were divided into eight molecular types
associated with distinct AFLP profiles. C.
neoformans may be classified into VNI/VNII genotypes corresponding to
AFLP1/1A/1B (serotype A), VNIII/AFLP3 (serotype AD), and VNIV/AFLP2 (serotype
D), while C. gattii may be
distinguished as VGI/AFLP4 (serotype B), VGII/AFLP6 (serotype B), VGIII/AFLP5
(serotype B or C), and VGIV/AFLP7 (serotype B or C). Interspecific hybrid
serotypes BD and AB correspond to AFLP8 and AFLP9, respectively [35, 37]. The
VNI and VGI genotypes are the most prevalent isolates of each species. The
Vancouver C. gattii isolates have a
VGII genotype that is further subtyped into VGIIa (major) and VGIIb (minor) [23]. A recent study employing MLST of one hundred and seventeen
isolates confirmed previous observations that four monophyletic lineages exist
within C. gattii. Based on these
findings, it was suggested that these lineages should be considered as separate
taxa, similar to the two monophyletic lineages within C. neoformans that correspond to varieties grubii and neoformans [45]. Another MLST study has indicated that although the Vancouver
Island C. gattii strains have
colonized a novel environment, they are not phylogenetically unique [46].


## 4. Ecology and Epidemiology ofC. gattii

C. gattii is endemic in tropical and subtropical regions such as Australia
where it is most commonly associated with eucalyptus trees, particularly Eucalyptus camaldulensis and Eucalyptus tereticornis [15, 47–49]. The
fungus has also been found to grow on other tree species such as almond (Prunus dulcis), golden shower (Cassia fistula), and Douglas fir (Pseudotsuga menziesii) in Colombia, Brazil, and Vancouver Island,
respectively [17, 47]. C. gattii has also been isolated from
insect frass in Australia and a wasp nest in Uruguay [50, 51].
Other species in the Filobasidiella lineage, such as Cryptococcus amylolentus, Tsuchiyaea
wingfieldii, and Bullera dendrophila,
have also been isolated from insect frass [52–54].  In this context, the recent discovery of C. gattii in the Pacific Northwest is
intriguing, since this temperate climate is characterized by mild and wet
winters and warm and dry summers. On Vancouver
Island, C. gattii has mainly been
found on trees in the coastal Douglas fir (CDF) biogeoclimatic zone such as
fir, cedar, and maple [23, 47]. The
fungus has also been isolated from the air, freshwater, seawater, and upper
layer of the soil of the BC mainland, the Gulf Islands, and Washington state [22, 23, 47].Ecological and
geographical differences exist among the different molecular types ofC. gattii. A survey conducted in 16
countries revealed that the molecular types most
commonly found in the clinic and environment were VGI and VGII [55]. Genotype VGIII has been found in the United States, Mexico, South America, Europe, India, Australia, and New
Zealand, while VGIV has been isolated in Mexico, Colombia, Europe, South Africa, and India [44, 55–58]. A recent epidemiological survey across Europe
showed that C. gattii clinical
infection is rare in this continent, represented by only six of 535 serotyped Cryptococcus isolates [59]. In this study, the genotypes of the six
European C. gattii isolates
identified were not determined. In Australia, the C. gattii VGI genotype is the most common clinical or environmental
isolate, while the VGII genotype is an infrequent cause of human or animal
infection [15, 50]. A notable exception to this overall pattern is the Northern
Territory of Australia that does not contain eucalyptus trees yet has the
highest incidence of clinical C. gattii infection. Interestingly, most human cases in this region are due to VGII,
suggesting that C. gattii has
colonized a new environmental niche [60, 61]. This
possibility is also supported by the fact that eucalyptus trees in the rest of
Australia have only yielded the VGI genotype [62].While the majority ofC.
gattii isolates in Australia's Northern Territory and on Vancouver Island
are of the VGII genotype, a notable difference between the two regions is the
remarkably limited diversity of the latter. Specifically, ninety five percent
of the environmental and clinical samples on Vancouver Island are of the VGII
genotype, and within this group 90% are of the major genotype VGIIa [47]. MLST studies have shown that the Vancouver Island VGIIa genotype
is identical to two known C. gattii strains: the 1975 Seattle human isolate NIH 444 (also known as CBS 6956 and
ATCC 32609) and the 1992 San Francisco environmental isolate CBS 7750 isolated
from E. camaldulensis [63]. These studies have also demonstrated that the VGIIa isolates from
Vancouver Island, the BC mainland, the Gulf Islands and northern Washington
State are genetically identical [22].
These findings indicate that the major genotype VGIIa has existed in the
Pacific Northwest for more than 30 years and it is hypothesized that VGIIa
originated from sexual mating between VGIIb and another unknown parental strain
[63]. Notably, VGI and VGII clinical isolates from Oregon differed from
the Vancouver Island VGI and VGII samples at one or more of the loci that were
analyzed [22]. The
reason for this is not clear but may be explained by divergent evolution from a
common genotype or the presence of distinct genetic isolates residing in the
Pacific Northwest. The VGIIb minor genotype on Vancouver Island is identical to
the unusually fertile Australian VGIIb clinical isolate NT-13, suggesting that
the Vancouver Island isolate originated in Australia [63]. The VGI genotype is rarely found on Vancouver Island and it
remains unclear whether it has actually colonized the environment, and the
molecular types VGIII and VGIV have not yet been reported [23, 46].
Recent studies on C. gattii clinical
and environmental isolates from South America have shown that VGII predominates
over the other molecular types in both Colombia and Brazil as observed on
Vancouver Island [47, 64, 65].
Meyer et al. have hypothesized that
the Vancouver Island outbreak isolates originated in South America based on the
finding of α mating-type cells within the Brazilian C. gattii VGII population and the fact that all Vancouver Island C. gattii isolates found to date have
also been of the α mating type [63, 65, 66]. In
addition, the VGII genotype has been present in Brazil longer than it has
been on Vancouver Island [65]. In
contrast, C. gattii VGII isolates (serotype B) in Colombia are mainly of
the opposite a mating type, although large numbers are present in regions that
have a temperate climate that is similar to Vancouver Island [64].


## 5. Virulence Factors

C. gattii and C. neoformans share many attributes that increase their ability to
invade and survive in a host organism [92]. The main virulence factors identified in C. gattii to date include an outer polysaccharide capsule, melanin,
mannitol, extracellular proteinase, products of the laccase pathway, superoxide
dismutase, phospholipases, urease, and the STE12α transcription factor (a
homologue of Saccharomyces cerevisiae STE12) that is present only in the α mating type (Table 1) [93]. Other properties of C.
gattii contribute to its infectivity such as its ability to grow at
physiological temperature [15], its tolerance of low pH and elevated salt levels [47], and its ability to switch capsular phenotype [91].Table 1
C. gattii virulence factors
and their functions.
FactorFunctionCapsule and its associated polysaccharidesEvasion of
phagocytosis [67]Reduction of antigen
presentation [68]Reduction of
cytokine production [69]Induction of
suppressor T-cells which inhibit cell-mediated immunity [70]Inhibition of T-cell
responses by GXM [31]Inhibition of leukocyte migration into inflammatory sites by GXM [31, 71]MelaninProtection against
UV radiation [72]Protection against
oxygen and nitrogen free radicals [73, 74]May contribute to central nervous system tropism [6]Contributes to
negative cellular charge [75]MannitolSuggested increase
in intracranial pressure [76]Protection against
stress [77]Protection against
oxygen free radicals [78]Extracellular proteaseProteolytic activity
[79]May contribute to
degradation of proteins involved in tissue integrity and host immunity [79–82]Products of laccase pathwayDiphenol oxidation [83]Synthesis of melanin
[84]Degradation of wood
lignin [15]Superoxide dismutaseProtection against
oxidative stress [85]Protection against
oxidative burst produced by immune effector cells [85]PhospholipasesTissue invasion via
degradation of mammalian membrane lipids and lung surfactant [15, 86]UreaseExact function is
unknown [87]May aid in transfer
ofCryptococcus to central nervous
system [88]STE1α transcription factor (in cells of α
mating type)Upregulation leads
to synthesis of diphenol oxidase (which is a laccase) [89, 90]Growth at physiological temperature (37C°)Survival and
persistence in the host [15]Tolerance of low pHSurvival and
persistence in the environment [47]Tolerance of elevated saltSurvival and
persistence in the environment [47]Phenotypic switchingChange in capsule
size—mucoid variant more virulent, smooth variant
suggested to be able to cross blood-brain barrier [91]A carbohydrate-rich
outer capsule that is composed primarily of glucuronoxylomannan (GXM) with
smaller proportions of galactoxylomannan (GalXM) and mannoproteins is the major
virulence factor for bothC. gattii and C. neoformans [94]. The capsule may change in composition and size through a process
called phenotypic switching (described further in the Notable Attributes
section) and induces suppression of the host immune response by various
mechanisms including the downregulation of cytokine and chemokine expression
in dendritic cells (Table 1) [91, 95].
Kinetic studies have shown that complement component C3 binds less efficiently
to C. gattii compared to C. neoformans, suggesting that C. gattii enhances virulence by
preferential evasion of immune recognition [96]. Species-specific variation in the expression of other virulence
factors may also play a role in their pattern of infectivity and organ
dissemination. For instance, it was observed that extracellular proteinase
production is lower in certain strains of C.
gattii compared to a number of C. neoformans isolates, suggesting
that C. gattii may less efficiently
degrade proteins involved in tissue integrity and host immunity such as
collagen, fibrin, complement, and immunoglobulin [79–82]. This
finding may explain why C. gattii lesions are often more circumscribed compared to C. neoformans, a characteristic that could also reduce local and systemic dissemination [82, 97]. Similarly, in vitro experiments with a variety of C. neoformans and C. gattii isolates from South America have
shown that C. neoformans has increased
urease production relative to C. gattii [98]. Intratracheal administration of the highly virulent C. neoformans H99 (serotype
A) to mice suggests that
urease may aid in the dissemination of the fungus from the lung to the central
nervous system, although the exact mechanism is unknown [88]. It is also interesting to note that virulence factors are involved not only in pathogenesis but also in
commensalism. For instance, C. gattii was observed to share an endophytic relationship with decaying wood of both
eucalypt and noneucalypt trees, where the laccase enzyme system appears to
play a role in digestion of lignin [15, 50].Given
thatC.
neoformans and C.
gattii appear to share many of the same virulence factors, it is intriguing
that C. gattii most commonly infects
immune competent individuals while C.
neoformans primarily infects the immune compromised host. One explanation
for this observation is that immune compromised individuals simply have more
environmental exposure to C. neoformans compared to C. gattii [5]. Furthermore, the contribution of host genetic background to
resistance against cryptococcal infection is even less well understood.
Interestingly, certain groups of individuals such as the Australian aboriginal
population may be predisposed to C.
gattii disease [62]. Modern molecular
dissection of a microorganism's virulence
factors may allow researchers to better understand the genetic mechanisms
underlying virulence. A recent example of this approach used systematic
targeted gene deletion in C. neoformans with comprehensive profiling of
individual mutants in an animal model [99]. A similar large-scale strategy may be required to clearly delineate the pathogenesis of C. gattii
and may in turn stimulate the development of novel therapeutic strategies.


## 6. Notable Attributes ofC. gattii

Phenotypic switching and same-sex mating are two interesting attributes that contribute to the virulence
ofC. gattii. The term phenotypic
switching refers to an adaptive mechanism characterized by structural
modifications of the extracellular capsule and cell wall [91]. This phenomenon occurs infrequently in vitro or during chronic infection and a switch to a more mucoid
form is associated with greater virulence in C. neoformans [91, 100].
Reversible phenotypic switching between a smooth
and mucoid variant of C. gattii strain NP1 has also been identified
[100].  Shortly after infection, the mucoid form of NP1 is most commonly
observed; however, a subsequent switch to the smooth form characterized by
reduced capsular polysaccharide allows for easier penetration of the
blood-brain barrier and dissemination to the brain [91]. Evaluation of survival and fungal burden in BALB/c mice following
intravenous or intratracheal infection with either C. gattii NP1 phenotypic variant demonstrated increased virulence
of the mucoid form. In this study, both mucoid and smooth variants were found
in the lung homogenates while only the smooth variant was found in the brain
homogenates. In terms of the immune response, infection with the smooth variant
elicited a greater inflammatory response as characterized by lymphocyte and
monocyte infiltration and gave rise to smaller cryptococcomas than the mucoid
variant [91].In contrast to most
AustralianC. gattii isolates, it was
demonstrated that all Vancouver Island isolates belong to the α mating-type and
are unusually fertile [63, 66]. The
mechanism by which cryptococcal cells of an identical mating type replicate is
not known. However, it has been suggested that C. gattii undergoes same-sex mating, a process that has been
studied in C. neoformans [63]. Upon nutrient limitation, α haploid cells can undergo sexual
recombination via fruiting, a process by which haploid cells fuse, chromosomal
reassortment and recombination occur, followed by meiosis and sporulation [66, 101].
Same-sex mating between two α cells (rather than an α and an a cell) may confer
a survival advantage and could explain why all Vancouver isolates are of the α
mating type [101]. Both traditional and same-sex mating in C. neoformans have been observed in the laboratory but not in
nature [63, 66, 101, 102].


## 7. Animal Models ofC. gattii Infection

A limited number of animal model studies ofC. gattii infection have been reported in literature. One group
has investigated the survival of A/J inbred mice following intranasal infection
with different Vancouver Island C. gattii molecular types. In this report, the major genotype VGIIa appeared most
virulent (20% survival at 15 days post-infection), the minor genotype VGIIb was
avirulent (100% survival at 55 days post-infection), and the VGI genotype was
similar in virulence to VGIIa [63]. These findings are consistent with recent clinical experience that
has shown VGIIa to be the most common isolate from patients [21, 22, 24, 103].Further
insights into disease pathogenesis have been derived through animal studies
usingC. gattii isolates that were
not obtained from Vancouver Island. One study compared mouse and human
pulmonary inflammatory responses to intratracheal infection with 10 different
Australian isolates of C. gattii [104]. In BALB/c mice, 6 isolates did not elicit any inflammatory
response, 3 provoked a minimal response, and 1 resulted in a strong host
inflammatory response that took several weeks to develop. The peak inflammatory
response was observed 5 weeks after infection and was characterized by foamy
macrophages, lymphocytes, poorly defined granulomas containing some giant cells
as well as C. gattii yeast, and
destruction of lung tissue. In humans, variable
pulmonary pathology has been detected including poorly formed granulomas
containing intracellular C. gattii,
lymphocytic interstitial pneumonitis, tissue necrosis, and fibrosis. In both
species the local lymphocyte pool consisted largely of T-cells with a 2:1 ratio
of CD4 to CD8 cells in humans [104].A second study examined the pathology seen in BALB/c mice following
systemic infection with the clinical isolateC. gattii 9714 (CBS 6996, serotype B, VGIII/AFLP5) under different
experimental conditions [105, 106]. It
was observed that both immune competent and hydrocortisone-treated mice
developed C. gattii disease,
contrasting with the apparent predilection of immune competent humans to this
infection. At high doses of C. gattii,
immune suppressed mice were more likely to develop severe disease compared to
immune competent mice. SCID mice that lack T- and B-lymphocytes were more
susceptible to infection relative to wild-type BALB/c, suggesting a protective
role for these cell types [105]. Comparative studies showed that C. neoformans 9759 (a serotype A clinical isolate) has increased virulence compared to C. gattii 9714 after systemic infection of BALB/c mice, regardless
of the immune state of the animal. Specifically, C. neoformans 9759 infected the brain and lungs of BALB/c mice,
while C. gattii 9714 affected the
lungs and skin. Ulcerative lesions on the tail due to intravenous C. gattii infection were more common in
immune competent mice, while rectal prolapse was more common in immune
suppressed mice. This study also demonstrated that infection by either C. neoformans 9759 or C. gattii 9714 may cause
gastrointestinal pathology, although its incidence was rare [105].A third study examined the pathogenesis of fiveCryptococcus isolates administered to BALB/c mice via the
intraperitoneal route [107]. The isolates included C.
gattii GR52 and GR56 (both serotype B), from immune competent goats that
died of pneumonia in Spain; C. gattii B4506
(serotype B), an Australian environmental isolate known to be highly pathogenic
in mice; C. gattii I-682 (serotype
C), from a Colombian native almond tree; and C. neoformans GR297 (serotype D), from an AIDS patient in Spain
with meningitis. Two of five mice infected with C. neoformans GR297 developed liver and peritoneal abscesses and
one mouse died after four weeks. Fungal cultures of spleen, liver, kidney,
testes, lung, and brain were done for each group and were positive in at least
one organ for GR52, GR297, GR56, B4506, and I-682 in 80%, 77%, 70%, 70%, and
33% of the cases, respectively [107]. Based on the frequency of cryptococcal
growth in the organ cultures, all fungal isolates with the exception of
the serotype C sample I-682 displayed similar virulence in BALB/c mice. Among
the organs tested, the spleen was most often positive for Cryptococcus (91%), followed by the liver (75%), kidney (75%),
testes (71%), lung (62%), and brain (29%). C.
gattii GR52, the cause of a pneumonia outbreak in goats, was also isolated
from the lungs of all BALB/c mice studied, suggesting that it has a particular
tropism for lung tissue. Pathologic findings associated with infection of the
brain included areas of spongiosis in the white matter with an abundance of
encapsulated yeasts in the basal ganglia while infected lungs showed
encapsulated yeasts within alveoli, peribronchial vessels, and interalveolar
spaces. Interestingly, inflammation was not observed in either organ among
infected mice [107].These studies show that the route of experimental cryptococcal
infection is a major determinant of the site of disease. In general,
intravenous infection directly induces systemic disease, whereas inhalation of
the fungus via the airway mimics the natural route of infection and leads to
primary disease in the lung [108]. For example, in the second study both the cutaneous and
gastrointestinal pathologies may have been a direct result of C. gattii administration through the
tail vein, and significant involvement of the visceral organs and peritoneum in
the third animal study may have resulted from intraperitoneal inoculation of
the fungus. Nonetheless, the tissue tropism observed in humans is similar to
that seen in these mouse models [13, 15, 21]. In
addition to the route of infection, these studies also confirm that other
variables such as the C. gattii isolate, infectious dose, and immune status of the host also play a role in
the virulence of the fungus and the severity of infection [15].


## 8. Veterinary Cases

C. gattii has the potential to infect a
wide range of animals throughout the world, including wild, farm, domestic, and
aquatic animals, along with a variety of birds. This broad host range was
observed during the recent outbreak in British Columbia [23, 109, 110]. In
order to study the epidemiology of veterinary cases, diagnostic methods to test
for infection in animals generally consist of serum and/or tissue sampling and
nasal swabbing [28, 109, 111–113]. A
recent study examined the characteristics of C. gattii infection in domestic animals in southwestern BC,
involving 78 feline and 51 canine cases [28]. It was observed that cats were 4.4 times more likely than dogs to
be positive for C. gattii infection,
and that all cases in dogs and 50% of cases in cats were due to C. gattii serotype B [111]. In cats, the median age at diagnosis was 7.3 years, whereas in
dogs it was 2.3 years [28]. The tissue tropism of C.
gattii disease differs slightly in cats and dogs. Among canines, the
affected organs included the respiratory system (52%), central nervous system
(42%), and the skin or gastrointestinal tract (6%), while in felines 56% were
respiratory, 26% involved the central nervous system, and 19% were dermal with
no cases of gastrointestinal involvement [28].Another study was
carried out on Vancouver Island to determine whether wild animals were infected
withC. gattii. Of the 91 animals
that underwent swabbing of the nasal cavity, two eastern grey squirrels were
found to be positive for C. gattii.
This number is similar to the incidence of infection in asymptomatic companion
animals; specifically 1.1% of dogs, 4.3% of cats and 1.5% of horses were
positive for C. gattii [109]. Subclinical C. gattii infection in dogs and cats does not necessarily progress to clinical disease.
In some cases it may be cleared while in other cases it may persist or develop
into more severe disease [115]. It has also been suggested that infected wild animals may be an
important vector for the spread of C.
gattii from Vancouver Island to geographic regions that do not harbor the
organism and that veterinary cases may also be a reliable sentinel of human
disease [109]. The latter assertion is based on the observation that C. gattii infection was identified in
animals prior to the human outbreak that began on Vancouver Island in 1999 and
that nearly twice as many animals appeared to be infected with C. gattii compared to humans between
1999 and 2003 [116]. These observations also highlight a potential role for animals in
transmission of infection to humans. While zoonotic transmission of C. gattii has not been reported to date,
transmission of C. neoformans from
pet birds to humans has been documented, and possible human-to-human
transmission has also been described [117–119]. In
addition to the veterinary cases in the Pacific Northwest, outbreaks of C.
gattii involving goats in Spain [120] and psittacine birds in Brazil [112] have also been observed.


## 9. HumanC. gattii Infection

C. gattii may cause mild to severe clinical disease in the apparently healthy
as well as immune compromised host. Like other cryptococcal species, C. gattii enters the human host through
the inhalation of airborne propagules and targets the lung as a primary site of
infection [22, 108]. In
some cases dissemination via the bloodstream may occur, most commonly to the
central nervous system (CNS) with occasional spread to other organs such as
skin, eye, and prostate [13, 15, 82]. The
predominant manifestations of C. gattii infection
involve the lungs in the tropical climate of Australia (66% pulmonary) as well
as the temperate climate of Vancouver Island and its surroundings (75%
pulmonary) [21, 60].A study performed
by the British Columbia (BC) Cryptococcal Working Group at the University of
British Columbia reviewed the clinical aspects of 171 individual human cases ofC. gattii infection on Vancouver
Island from 1999 to 2006 [21]. It was determined that the mean age at diagnosis was 59 years,
with a range of 2 to 92 years. Males (56%) appeared to be slightly more
susceptible to infection than females (44%) [21]. The clinical patterns of C.
gattii infection were pulmonary (75%), neurological (8%), combined (9%),
and unknown (8%). Of those patients with combined disease, the main sites
involved the lung and CNS, and less commonly the skin and lung, or skin and
CNS. Death was a rare outcome of C.
gattii infection on Vancouver Island with 8 fatalities from 1999 to 2006,
or approximately one death per year. Of the deceased patients, five presented
with both pulmonary and CNS pathology, three had other underlying
comorbidities, and one had an adverse reaction to therapy. The mean age of the
deceased patients was 61 years (range from 26 to 87) [21].A summary of the
most recent case reports associated with the Pacific west coast is presented in
Table2. Among the first 8 patients, three patients resided in British
Columbia, two patients resided in Oregon State, one resided in Washington
state, one resided in Alberta, and one patient was a tourist from Denmark
visiting Vancouver Island [22, 24, 25, 103]. The
place of residence of patients 9 to 12 was not specified, although they
represented patients discharged from Vancouver Island hospitals [114]. Six of the cases were either not directly exposed to C. gattii on Vancouver Island or were
exposed many years earlier. This suggests that another source of C. gattii may exist outside of Vancouver
Island, or that the fungus may have been transiently present in other
geographic regions due to dispersal mechanisms [22, 121].
Also, the fact that in two cases
infected individuals had traveled to Vancouver Island 4
and 14 years before developing symptoms raises the possibility that C. gattii may remain latent in the body
and reactivate at a later time [5].Table 2
Summary of case
reports ofC. gattii infection.
Age (sex)ResidenceExposureHealth conditionsSymptomsPathology (molecular type)1 [22]47 (M)BC mainland (on coast north
of VI)Yard/landscaping work at
residenceChronic hepatitis C, drug
addictionCough, chills, night sweats,
nausea, muscular pain, headache, appetite loss, stiff neckLung/brain nodules (VGI)2 [22]48 (F)BC (lower mainland)Deforestation near
residence, yard work, last visited VI 4 years agoNoneDyspnea, fever, chills,
headache, night sweats, appetite loss, nausea, muscular painLung mass (VGIIa)3 [22]73 (F)BCLittle outdoor exposure,
last visited VI 14 years agoChronic renal failure, lung
disease, breast cancer, hip surgeryNoneLung nodule
(VGIIa)4 [22]59 (M)ORNo travel out of OR in last
yearScarred lung tissue (due to
occupation)Cough, dyspnea, fever,
chills, nausea, weight loss, muscular painNot specified (VGIIa)5 [22]87 (M)ORTravel to parts of OR, WA
& COChronic lymphocytic leukemia
(oral steroid)Fever, weight loss, appetite
lossMeningitis (VGIIb)6 [24]74 (M)WA (Puget Sound)Travel to south CA and HI in
last yearLarge granular lymphocytic
leukemia (steroid and hormone)CoughLung nodule
(VGIIa)7 [25]45 (F)AlbertaHolidays spent on southeast
shore of VI, park visitsNoneHeadache, blurred vision,
photosensitivity, nausea, vomiting, coughLung opacities, airspace
disease, meningitis
(not specified)8 [103]51 (M)DenmarkTravel to Victoria and VI
east coast for 7 days, garden visitsPsoriatic gout
(nonsteroidal anti-inflammatory drug)Chest pain, fever, cough,
dyspnea3 large nodular infiltrates (VGIIa)9 [114]54 (M)Not specifiedNot specifiedBradycardia, acute
myocardial infarction, smokerNoneLung opacity
(not specified)10 [114]62 (M)Not specifiedNot specifiedPrevious smokerChest painLung nodules (not specified)11 [114]69 (F)Not specifiedExposure to tuberculosisPrevious smoker, total
colectomy due to ulcerative colitisCough, dyspneaCavitary lesion
(not specified)12 [114]65 (M)Not specifiedOccupational (painter)Previous smoker, mild
recurrent hemoptysisNot specifiedLung opacity, meningitis
(not specified)Legend: BC: British
Columbia, CA: California, CO: Colorado, HI: Hawaii, OR: Oregon, VI: Vancouver
Island, WA: Washington.In the BC
Cryptococcal Working Group study, the pulmonary symptoms most commonly observed
in the 171 cases were cough and dyspnea, while the main symptom involving the
CNS was headache [21]. Similarly, the usual symptoms in the Pacific west coast case
reports were cough and shortness of breath, although some patients developed
nausea, fever, headache, muscle pain, and loss of appetite. It is important to
recognize that the clinical presentation of C. gattii infection may be
quite subtle. For example, patients 3 and 9 were asymptomatic while patient 6
experienced only a nonspecific cough [22, 24, 114]. The
most common pathology observed in these patients was nodules of the lung in the
form of cryptococcomas. Interestingly, patient 7 presented with pulmonary
ground-glass opacities, a pattern that is quite distinct from circumscribed
nodules and rarely observed in the clinic [25]. The specific pulmonary pathology for patient 4 was not reported [22].
Meanwhile, CNS pathology was documented in four patients: patient 1 had
cerebral cryptococcomas and patients 5, 7, and 12 presented with meningitis [22, 25, 114].All 171C. gattii cases from the BC Cryptococcal
Working Group study were caused by C.
gattii serotype B [21]. This finding is consistent with the fact that serotype B exists at
a much higher frequency in the environment compared to serotype C. In fact, the
identification of C. gattii serotype
C in the environment had not been described prior to 1998 [50, 122]. It
also appears that the C. gattii major
genotype VGIIa is highly associated with human disease: 82% of the cases were
VGIIa, while the others were VGIIb (9%) and VGI (6%) [21]. Similarly, 5 of the 12 case reports from the Pacific west coast
were VGIIa, one was VGIIb, and another was VGI, while the rest were
unspecified. Apart from place of residence or travel, the main risk factors
associated with the development of C.
gattii disease were smoking (50%), oral steroid use (30%), invasive cancer
(24%), and chronic lung disease (13%). HIV infection (4%) and receipt of an
organ transplant (3%) were considered less common risk factors for the
development of C. gattii disease in
this study population [21]. Examination of the case reports shows a similar pattern; 4 of the
12 cases had a current or past history of smoking, and 7 of the 12 patients
suffered from chronic disease and/or cancer, had undergone recent surgery, or
had been exposed to corticosteroids.Thus far, the
median incubation time ofC. gattii on Vancouver Island has been estimated to be 6-7 months with a range from 2 to 11
months [123]. One exception is the relatively short incubation period of six
weeks that was observed in the Danish tourist visiting Vancouver Island [103]. The potentially long time frame from exposure to symptoms may
impede the diagnosis of C. gattii infection.
It is likely that the clinical incubation period depends on several variables
such as differences in host immunity and the intensity of C. gattii exposure.


## 10. Microbiology

Laboratory testing
is essential to diagnoseC. gattii infection and generally requires analysis of tissue or fluids from infected
sites such as cerebrospinal fluid, bronchial washings, blood, and urine [12]. Light microscopy is an efficient method for the rapid diagnosis of
cryptococcosis. For this technique, fluid samples are usually stained with
India ink while tissue samples may be stained with hematoxylin and eosin
(H&E), mucicarmine, or other stains [12, 20, 91]. In
both cases, cryptococcal cells appear round to oval in shape, surrounded by a
wide capsule. Infrequently, one may visualize nonencapsulated cryptococci
under the microscope [12]. In the laboratory, both C.
neoformans and C. gattii readily
grow as round cream-colored mucoid colonies on Sabouraud dextrose agar, a
selective medium that is widely used for the isolation of yeast [12]. Alternatively, malt extract agar may be used to selectively
isolate fungi [12, 47].
Differential microbial media such as Staib agar or birdseed agar may be used to
distinguish dark brown colonies of C.
neoformans or C. gattii from
other fungi [47, 124].
Further differentiation of cryptococcal species can be accomplished on
L-canavanine-glycine-bromthymol blue (CGB) agar; C. gattii colours the medium blue while C. neoformans does not (medium remains yellow) [125]. Though not required for clinical management, the fungus may be
viewed in its sexual state by growing the cells on V8 medium in the dark
followed by fixation and staining to visualize the hyphae, nuclei, and septa [101]. Finally, it is possible to determine the exact serotype of a
cryptococcal isolate using a slide agglutination assay although commercial kits
for this purpose are not currently available [22, 25].In vitro activity of antifungal agents
may be helpful in guiding the clinical management of severe cryptococcal
infection including meningoencephalitis [126]. A slower clinical response of C. gattii to antifungal
therapy has been attributed to serotype-specific differences in antifungal
activity though there are no consistent or predictable data to support this
hypothesis [127, 128]. To
evaluate this possibility further, a recently published study compared the
susceptibility of 86 C. neoformans and 42 C. gattii isolates to
various antifungal drugs including amphotericin B, flucytosine, fluconazole,
posaconazole, voriconazole, and isavuconazole [129]. The major finding was that all antifungal agents tested retained
activity against all cryptococcal isolates with the newer azoles exhibiting
greater potency compared to fluconazole and flucytosine. In this report, no
significant differences in drug potency were observed for C. gattii serotypes
B or C compared to other serotypes.


## 11. Spread of the Vancouver IslandC. gattii Outbreak

The colonization ofC. gattii on Vancouver Island, and
possibly other adjacent regions, indicates that this fungus has the ability to
adapt to new environmental conditions. It has been suggested by Kidd et al. that the temperate climate of
Vancouver Island may provide a favorable niche for the survival and dispersal
of C. gattii [23]. Global warming has also been proposed to favor C. gattii colonization of new geographic
regions [23]. Studies in Colombia have also suggested a correlation between
climate and distribution of cryptococcal serotypes A to C. Specifically,
differential tolerance of climatic and possibly other environmental conditions
by individual serotypes may affect their geographic and ecological distribution
[37, 130]. The
modes of dispersal of C. gattii have
been studied on Vancouver Island. Some trees in the region show intermittent
positivity, suggesting the movement of fungal spores [121]. High C. gattii levels
exist in public areas such as park entrances, parking lots, and beaches despite
the fact that these areas are characterized by low tree density [47, 121]. A
significant amount of C. gattii dispersal appears to be mediated by human activity, including contact with
footwear and car wheel wells, deforestation, and gardening [121]. Avian and perhaps other animal migrations, as well as insect vectors such as beetles and
caterpillars [15], may also contribute to dispersal [121].HasC. gattii colonized other regions on the
Pacific west coast? It is known that C.
gattii (serotype C) was isolated from a eucalyptus tree in San Francisco in
1992, but since then C. gattii has
not been found in that environment. In order to determine whether C. gattii has spread to the United
States, Fraser et al. investigated
possible colonization on the San Juan Islands, the closest U.S. region to
Vancouver Island [131]. The San Juan Islands also exhibit a temperate climate zone that is
similar, but not identical, to the CDF biogeoclimatic zone of Vancouver Island.
Although other fungi of the phylum Basidiomycota,
namely Cryptococcuslaurentii and Cryptococcus cellulolyticus, were isolated, C. gattii was not detected. This suggests that C. gattii may not have yet colonized the San Juan Islands, or
dispersal of the fungus is impeded by a geographical barrier that is the Juan
de Fuca strait [131]. In a recent study by MacDougall et al., C. gattii was
isolated from environments outside of Vancouver Island including the British
Columbia mainland, the Gulf islands, and Washington state, but no positive
environmental samples were found in Oregon [22]. In
the clinic, C. gattii has been
isolated in Seattle (1975),
Vancouver Island, and the British Columbia mainland, and most recently in
Oregon, Washington, and Alberta [22–25, 63].


## 12. Conclusion

The recent outbreak ofC.
gattii infection in the Pacific Northwest highlights the fact that this fungus
is an important emerging pathogen that can adapt to new potential environmental
niches. C. gattii isolates found on
Vancouver Island appear to be hypervirulent, as the number of infected
individuals per million people per year between 2002 and 2005 was more than 36
times higher on Vancouver Island compared to endemic regions such as Australia [22, 23]. It is in the interest of scientists and clinicians alike to better
understand the pathogenesis of C. gattii disease in order to discover effective prevention and treatment strategies,
including measures to limit human exposure to this pathogenic fungus. Employing C. neoformans as a model organism to
understand the disease-causing potential of C.
gattii is no longer sufficient since fundamental disparities such as
differences in host tropism and the expression of certain virulence factors
exist between the two species.



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*Source: 840452-2009-05-25.xml*