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CHAPTER 12

Cryptococcus species
Catriona L. Halliday and Sarah E. Kidd

Introduction to Cryptococcus C. neoformans

The basidiomycete genus Cryptococcus comprises a diverse range
of over 70 species characterized by round or oval yeast-​like cells
that reproduce by narrow-​necked budding. Cultures are generally
mucoid on solid media and most species are encapsulated. All spe-
cies produce urease and are non-​fermentative (Howell et al. 2015).
Taxonomy
Cryptococcus neoformans and Cryptococcus gattii are the princi-
pal pathogenic species and the causative agents of cryptococcosis.
C. neoformans is currently recognized as a species complex com-
prising the two varieties var. grubii (serotype A) and var. neofor-
mans (serotype D). C. gattii has two serotypes, B and C, and was
recognized as a distinct species from C. neoformans in 2002 (Kwon-​
Chung et al. 2002). Other cryptococcal species, such as C. adeliensis,
C. albidus, C. curvatus, C. flavescens, C. laurentii, and C. uniguttula-
tus, were traditionally considered to be non-​pathogenic, but occa-
sionally cause invasive infections in humans (Khawcharoenporn
et al. 2007).
C. neoformans var. grubii has a worldwide distribution and causes
95% of all C. neoformans infections (Ma and May 2009). It has been
isolated from a variety of environmental sources, including avian
excreta (particularly pigeons), soil, and decaying wood from trees.
C.  neoformans var. neoformans infections are most prevalent in
Europe, where it accounts for 30% of isolates (Ma and May 2009).
The overwhelming majority of symptomatic C.  neoformans
infections occur in patients with impaired cell-​mediated immu-
nity, including those with advanced HIV (human immunodefi-
ciency virus) infection, organ transplantation, haematological
malignancies, and diabetes mellitus; infections among those with
intact immune systems are rare. Meningitis is the predominant
clinical presentation, with an estimated incidence of 1 million cases
per year, with most cases occurring in sub-​Saharan Africa (Park
et  al. 2009). After candidiasis and aspergillosis, cryptococcosis is
the third most common invasive fungal infection in patients who
undergo solid organ transplantation, particularly kidney and liver
transplant recipients (Pfaller and Diekema 2010).

Pathogenesis
Irrespective of the species, cryptococcal infections are acquired
following inhalation of the infectious propagule from the envir-
onment. In most cases, symptoms do not develop, indicating that
most immunocompetent people clear the infection. Infections
may be localized or disseminate, often to the central nervous
system (CNS). Dissemination may follow reactivation of dor-
mant infection, particularly during immunosuppression. Disease
severity is dependent on both fungal virulence factors and the
host’s immune response. Although not as comprehensively stud-
ied, transmission, virulence factors, and immune response to
other species of Cryptococcus resemble those of C.  neoformans
and C. gattii (Table 12.1) (Ma and May 2009).
(See Chapter  8 for further details of the virulence factors of
Cryptococcus.)
Epidemiology
Cryptococcosis occurs in both humans and animals, including
domestic dogs and cats, and native Australian animals such as
the koala. Animal-​to-​human and human-​to-​human transmission
has been documented rarely. The disease is uncommon in chil-
dren, with a prevalence of 1% in children with AIDS (Pfaller and
Diekema 2010).
C. gattii
C.  gattii has a comparatively restricted geographical distribution,
occurring predominantly in tropical and subtropical climates such
as Australia. Its prevalence in temperate climates is uncommon,
although an emergence on Vancouver Island, Canada, was identi-
fied in 2002, with subsequent expansion to the Pacific Northwest
and other areas of the USA (Pfaller and Diekema 2010). C.  gat-
tii has a specific ecological association with numerous species of
Eucalyptus trees, which although native to Australia, have been
extensively exported to other tropical climates. The Canadian iso-
lates are associated with a range of native non-​Eucalyptus species
such as the Coastal Douglas Fir (Kidd et al. 2007).
Historically considered a pathogen in persons with apparently
normal immune systems, a recent review in Australia noted an
increase in HIV-​negative immunocompromised patients due to
cancer, idiopathic CD4 lymphopenia, and long-​term immunosup-
pressive therapies (Chen et  al. 2012). Similarly, the emergence in
Canada and the Pacific Northwest, USA, identified new risk factors
for infection, including HIV/​AIDS, cancer, and smoking (Harris
et al. 2011).
C.  gattii causes only 1% of cryptococcosis cases worldwide. In
Australia’s Northern Territory, where C. gattii infection is endemic,
the annual incidence is 6.5 cases per million (Chen et al. 2012); how-
ever, in the recent Pacific Northwest outbreak the annual incidence
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Table 12.1  Virulence factors of Cryptococcus species
Virulence factor Role
Polysaccharide capsule: Inhibits phagocytosis and protects against
glucuronoxylomannan dehydration
(GXM) and
glucuronoxylomannogalactan
(GXMGal)
Melanin Protects from UV light and environmental
conditions; an antioxidant protecting against
immune response and antifungal drugs
Laccase Catalyses production of melanin; helps to
establish an ecological niche in rotting wood.
Activity lower in non-​neoformans/​gattii species
Proteinases and Degrades host cell membrane and lyses cells,
phospholipases providing nutrients for the organism and
protection from the host
Urease Catalyses hydrolysis of urea to ammonia
and carbamate; facilitates blood-​to-​brain
transmission
α mating type More prevalent and more virulent than
a mating type
was as high as 35 cases per million (Pfaller and Diekema 2010), and
in northern Brazil, C. gattii accounts for 62% of all cryptococcosis
cases (Ma and May 2009). Cryptococcosis caused by this species
is characterized by the development of cryptococcomas (large
mass lesions) in the lung and brain. Morbidity from neurological
disease is high, although mortality rates are lower compared with
C. neoformans infections.
Other Cryptococcus species
Non-​neoformans/​gattii species are widely distributed geographically
and have been identified from air, soil, water, pigeon droppings, and
food. Some species, such as C.  laurentii and C.  uniguttulatus, can
colonize humans (Khawcharoenporn et  al. 2007). Eighty per cent
of the non-​neoformans/​gattii infections are due to C.  albidus and
C. laurentii. Impaired cellular immunity is the most common risk
factor, with HIV infection and low CD4 counts a common comor-
bidity. For C. laurentii infection, invasive devices are an additional
risk factor (Khawcharoenporn et al. 2007; Arendrup et al. 2013).
Clinical presentation
Cryptococcal infections may present acutely or chronically, with
clinical presentations varying depending on the stage of disease
rather than the causative species. The most common clinical mani-
festations involve CNS, bloodstream, and pulmonary infections
(Khawcharoenporn et al. 2007; Ma and May 2009) (see Chapters 22,
25, and 30).
Diagnosis
Within tissue sections, mucicarmine or Alcian blue stains the
capsule of Cryptococcus species to distinguish it from other yeasts
with similar morphologies. Cryptococcal cells are typically rounder
than the cells of Candida species and reproduce by narrow-​necked
Chapter 12  Cryptococcus species 81
Figure 12.1  India ink stain of Cryptococcus gattii cell in cerebrospinal fluid.
Approximate ×400 magnification.
Image reproduced courtesy of Catriona Halliday.
budding. Immunohistochemical staining allows identification and
discrimination of C.  neoformans var. grubii from var. neoformans
and C. gattii in formalin-​fixed tissues (Krockenberger et al. 2001).
For direct examination of cerebrospinal fluid (CSF), urine, and
other body fluids, the India ink stain can be used to visualize the
capsule, demonstrating a clear halo around the yeast under bright
field illumination (Figure 12.1).
Several commercial cryptococcal antigen tests are available to
detect capsular proteins of C.  neoformans and C.  gattii in serum
and CSF with high sensitivity and specificity. False positives have
been reported, and they are not a reliable diagnostic test for non-​
neoformans/​gattii species, despite having capsular antigens in com-
mon (Howell et  al. 2015). The IMMY Cryptococcal Lateral Flow
assay (Immuno-​Mycologics, Inc., OK, USA) is a point-​of-​care test
developed for the early diagnosis of cryptococcosis from serum,
CSF, and urine samples, which has replaced conventional antigen
techniques in many diagnostic laboratories. Its performance has not
been evaluated for the detection of non-​neoformans/​gattii infections.
Cultures of C. gattii can be differentiated from C. neoformans by
growth on CGB (canavanine-​glycine-​bromothymol blue) agar, turn-
ing the medium blue. C. neoformans does not grow on this medium.
Commercially available carbohydrate assimilation kits used to be
the mainstay of yeast identification; however, reliable species-​level
identification, particularly of unusual species, increasingly requires
rDNA sequencing of the internal transcribed spacer regions
and/​or D1/​D2 domains of the large subunit. MALDI-​TOF-​MS
(matrix-​assisted laser desorption/​ionization–​time-​of-​flight mass
spectrometry) can provide reliable species-​and subspecies-​level
identification of Cryptococcus species, but its accuracy depends on
database quality (Arendrup et al. 2013).
Treatment
All Cryptococcus species are intrinsically resistant to echinocandins.
Infections due to C. neoformans and C. gattii are initially treated with
amphotericin B, with or without flucytosine (5-​fluorocytosine), fol-
lowed by maintenance therapy with fluconazole, sometimes for life (see
Chapters 33 and 49). The emergence of resistance to amphotericin B,
82
82 Section 2   medically important fungi
flucytosine, fluconazole, or itraconazole has been reported, par-
ticularly after prolonged treatment or prophylaxis with fluconazole.
Untreated cryptococcal meningitis is fatal. However, appropriate anti-
fungal treatment, coupled with highly active antiretroviral therapy, has
greatly improved the prognosis of AIDS-​associated CNS cryptococ-
cosis (Pfaller and Diekema 2010) (see Chapter 33).
Optimal treatment strategies for infections due to other
Cryptococcus species have not been determined, but depend on the
site of infection. In vitro susceptibility testing indicates that C. albi-
dus, C.  curvatus, and C.  laurentii are susceptible to amphotericin
B, but less susceptible to flucytosine, fluconazole, and other azoles
than C. neoformans. Initial induction therapy with amphotericin B
until evidence of clinical improvement—​followed by step-​down to
fluconazole therapy if the isolate is susceptible—​is considered an
appropriate treatment regimen (Arendrup et al. 2013). Spontaneous
recovery in less severe cases of non-​neoformans/​gattii infections
with non-​CNS involvement may occur, usually as a result of cath-
eter or infected-​tissue removal (Khawcharoenporn et al. 2007).
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