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Introduction
Cryptococcus neoformans (Sanfelice) Vuillemin, the clas-
sical etiologic agent of cryptococcosis, is currently recog-
nized as a species complex, comprising C. neoformans
var. grubii, serotype A, C. neoformans var. neoformans,
serotype D, and C. gattii, serotypes B and C (Kwon-Chung
et al., 2011, Simwami et al., 2011). Cryptococcus neofor-
mans and C. gattii dier signicantly in their geographic
distribution and ecologic niches. A vast majority of cryp-
tococcal infections, particularly in immunocompromised
patients, are caused by C. neoformans var. grubii whereas
C. gattii accounts for a smaller proportion of cases but it
frequently infects immunocompetent patients in tropical
and subtropical regions. Cryptococcus neoformans is a life-
threatening etiologic agent of fungal meningitis, with an
increasing number of global cases occurring in HIV/AIDS
patients but more so in developing countries. An estimated
one million cases of cryptococcal meningitis occur glob-
ally per year in AIDS patients, resulting in approximately
625,000 deaths (Park et al., 2009). India has the second
largest burden of cryptococcosis due to an estimated
population of 3.1 million to 9.4 million persons living with
HIV/AIDS (UNAIDS, 2006). e country has a documented
high prevalence (1.7–4.7%) of cryptococcosis in persons
with HIV/AIDS (Kumarasamy et al., 2003; Vajpayee et al.,
2003). e large global burden of cryptococcosis presents
a number of challenges to public health, particularly in
resource-decient regions of high HIV prevalence in sub-
Saharan Africa and South/Southeast Asia. Multiple strate-
gies, including environmental surveillance, increase in the
number of diagnostic laboratories and strengthing of their
infrastructure are required to combat the increasing threat
to public health.
In the past decade, a more virulent genotype of
C. gattii, AFLP6A/VGIIa; AFLP6C/VGIIc, has emerged as
REVIEW ARTICLE
Environmental prevalence of Cryptococcus neoformans and
Cryptococcus gattii in India: An update
Anuradha Chowdhary1, Harbans S. Randhawa1, Anupam Prakash1, and Jacques F. Meis2,3
1Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India, 2Department of
Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, e Netherlands, and 3Department of
Medical Microbiology and Infectious Diseases, Nijmegen, e Netherlands
Abstract
An overview of work done to-date in India on environmental prevalence, population structure, seasonal variations
and antifungal susceptibility of Cryptococcus neoformans and Cryptococcus gattii is presented. The primary ecologic
niche of both pathogens is decayed wood in trunk hollows of a wide spectrum of host trees, representing 18 species.
Overall, C. neoformans showed a higher environmental prevalence than that of C. gattii which was not found in the
avian habitats. Apart from their arboreal habitat, both species were demonstrated in soil and air in close vicinity of their
tree hosts. In addition, C. neoformans showed a strong association with desiccated avian excreta. An overwhelming
number of C. neoformans strains belonged to genotype AFLP1/VNI, var. grubii (serotype A), whereas C. gattii strains
were genotype AFLP4/VGI, serotype B. All of the environmental strains of C. neoformans and C. gattii were mating type
α (MATα). Contrary to the Australian experience, Eucalyptus trees were among the epidemiologically least important
and, therefore, the hypothesis of global spread of C. gattii through Australian export of infected Eucalyptus seeds
is rebutted. Reference is made to long-term colonization of an abandoned, old timber beam of sal wood (Shorea
robusta) by a melanin positive (Mel+) variant of Cryptococcus laurentii that was pathogenic to laboratory mice.
Keywords: Cryptococcus neoformans, Cryptococcus gattii, natural habitat, ecological niche, decaying wood, India
Address for Correspondence: Jacques F. Meis, Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital,
PO Box 9015, 6500GS Nijmegen, e Netherlands
(Received 15 June 2011; revised 15 July 2011; accepted 15 July 2011)
Critical Reviews in Microbiology, 2012; 38(1): 1–16
© 2012 Informa Healthcare USA, Inc.
ISSN 1040-841X print/ISSN 1549-7828 online
DOI: 10.3109/1040841X.2011.606426
Critical Reviews in Microbiology
2012
38
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16
15 June 2011
15 July 2011
15 July 2011
1040-841X
1549-7828
© 2012 Informa Healthcare USA, Inc.
10.3109/1040841X.2011.606426
BMCB
606426
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2 A. Chowdhary et al.
Critical Reviews in Microbiology
a primary pathogen on Vancouver Island and its adjoin-
ing areas in Canada and the USA, revealing extension of
this pathogen’s geographical domain to the temperate
climate (Kidd et al., 2004; Datta et al., 2009; Byrnes et al.,
2010). e new genotype has been responsible for over
350 cases of human cryptococcosis, 19 of which were fatal
despite aggressive antifungal therapy, and the outbreak
is still ongoing (Bartlett et al., 2008; Galanis et al., 2010;
Mak et al., 2010). It is noteworthy that the source of this
cryptococcal outbreak was traced to extensive coloniza-
tion of several native trees by C. gattii and its occurrence
in soil in the aected temperate region (Kidd et al., 2004,
2007b; Byrnes et al., 2010).
Cryptococcus neoformans and C. gattii are free-
living saprobes in nature and they infect their human
and animal hosts when air-borne infectious propagules
(yeast cells or basidiospores) are inhaled. As the infec-
tion is acquired from exogenous sources and it is ordi-
narily not transmissible from one infected individual
to another, it is vitally important to have rst-hand
knowledge of environmental prevalence of C. neofor-
mans and C. gattii with a view to designing any pos-
sible control measures against cryptococcosis. Besides,
it is necessary to make environmental isolations of
C. neoformans and C. gattii in order to probe their
genetic structure. Furthermore, the advances made
in genotyping techniques and their application to
environmental isolates will address some important
questions, such as, is there any evidence of ecologi-
cal specialization among C. neoformans isolates from
pigeon guano and those from decayed wood or other
plant debris? is paper aims to present an overview,
in global context, of the work done to-date on the envi-
ronmental prevalence of C. gattii and C. neoformans in
India and indicate the gaps in our existing knowledge.
Historical
Cryptococcus neoformans was isolated for the rst time
in 1894 by Sanfelice from peach juice in Italy (Sanfelice,
1894). He described it as an encapsulated yeast, dem-
onstrated its pathogenicity for laboratory animals and
named it Saccharomyces neoformans. In the same year,
Busse (1894) and Buschke (1895) in Germany reported
the rst clinical human case which they described as
Saccharomycosis hominis. For the following 57 years, this
pathogenic yeast was known only from clinical cases,
until the globally renowned medical mycologist, Chester
Emmons (1951) reported 4 incidental isolations of
C. neoformans in the USA during an investigation of 716
soil samples for Histoplasma capsulatum, employing the
mouse-inoculation technique. Subsequently, Emmons
(1955) reported frequent isolations of virulent C. neo-
formans strains from pigeon nests bearing old excreta,
indicating that it was an environmental reservoir for the
pathogen. His pioneering work stimulated worldwide
studies which demonstrated that desiccated pigeon and
other avian fecal matter constituted the most important
natural habitat of C. neoformans. Fritz Staib and cowork-
ers in Germany refocused attention on the pathogen’s
original isolation from peach juice by Sanfelice when
they reported its isolation from a ripe peach fruit (Staib
et al., 1973; Staib et al., 1974) and demonstrated in vitro
colonization by C. neoformans of dried leaves, stems
and other parts of various plants under dened labora-
tory conditions (Staib et al., 1972a; Staib et al., 1972b).
In 1986, C. neoformans was isolated from wood samples
collected from a hollow tree trunk inside an aviary in the
Zoological Garden, Antwerp, Belgium (Bauwens et al.,
1986). In this paper, reference was made to unpublished
observations of Daniëlle Swinne on the isolation of
C. neoformans var. neoformans from saw dust of tropical
trees, Entandophragma species, in a sawmill in Kinshasa,
Congo. It was stated that although the isolation of
C. neoformans from bark and wood from aviaries could
be due to contamination with bird droppings, it was nev-
ertheless possible that some trees could provide a natural
habitat for C. neoformans. Further investigations on the
role of wood in the natural history of C. neoformans was,
therefore, suggested.
e environmental niche of C. gattii remained
unknown for nearly two decades after this taxon was
described by Vanbreuseghem and Takashio (1970).
Attention to its natural habitat in trees was rst drawn by
Ellis and Pfeier (1990a) who isolated serotype B strains
from debris of leaves and owers of Eucalyptus camaldu-
lensis in Australia.
Isolation techniques
Sample collection
Apart from air sampling, the wide variety of environ-
mental samples that have been investigated include soil,
avian excreta, decayed wood and bark of trees or other
plant debris (Lazéra et al., 1993; Randhawa et al., 2001;
Kidd et al., 2007a; Kidd et al., 2007b; Illnait-Zaragozi
et al., 2010a). Samples of soil, plant debris or bark from
tree trunks are collected in clean, self-sealing polyeth-
ylene bags or screw-capped, 2-oz, glass bottles, using
metal spatula and forceps after cleaning them with 70%
ethanol. Decayed wood from inside tree trunk hollows is
scrapped with a sterile surgical scalpel and shavings or
small pieces of decayed wood are collected (Randhawa
et al., 2005). Aerial sampling may be done by exposure
of petri-dishes containing simplied Staib’s niger seed
medium, or using air samplers (Staib, 1962; Paliwal
and Randhawa, 1978; Randhawa et al., 2006; Kidd et al.,
2007b). Currently, a more ecacious sampling tech-
nique of swabbing is employed which is briey described
below.
Swabbing
Cotton-tipped swabs can be prepared in-house from
bamboo broom sticks, measuring 40-cm-long and 2 mm
in thickness. e swab sticks are wrapped in craft paper
and sterilized by autoclaving. Sampling of decayed wood,
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Environmental prevalence of C. neoformans and C. gattii in India 3
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bark and plant debris is done by rubbing the swabs moist-
ened with sterile physiological saline, containing gen-
tamycin (25 mg/L). e swab handles are then cut short
and transferred to sterile, screw-capped glass bottles
(75 × 25 mm) and brought to the laboratory for processing.
e swabs are inoculated directly or after suitable dilu-
tion on simplied Staib’s niger seed plates. e ecacy of
swabbing versus a conventional technique for sampling
of decayed wood in tree trunk hollows for isolation of
C. neoformans and C. gattii was evaluated by Randhawa
et al. (2005). Of 42 known positive wood samples, swab-
bing was successful for isolation of C. neoformans in 40
(95%) as against 32 (76%) by the conventional technique,
and this dierence was statistically signicant (p < 0.01).
Moreover, the conventional technique yielded 24% false-
negative results in striking contrast to only 5% by swab-
bing. Besides, swabbing yielded a signicantly higher
C. neoformans mean colony count per positive sample
than did the conventional technique (p < 0.005), thus
demonstrating the superiority of the former technique.
Notably, application of the swabbing technique revealed
that over 80% of the S. cumini trees in one locality of
Delhi harboured the C. neoformans species complex in
decayed wood inside their trunk hollows. In view of its
greater ecacy, the swabbing technique has been widely
adopted in environmental studies.
Sample processing
Floatation-sedimentation technique
Sample processing is done by a oatation-sedimen-
tation technique, previously used for isolation from
soil of Coccidioides immitis, the etiologic agent of coc-
cidioidomycosis (Stewart and Meyer, 1932). Briey, a
measured quantity of the test soil samples, avian excreta,
plant debris, etc. is suspended in sterile physiological
saline and mixed thoroughly by vortexing. e suspen-
sion is allowed to stand for sedimentation, followed by
separation of the supernatant.
Direct inoculation
Measured aliquots of the supernatant are directly inocu-
lated on plates of simplied Staib’s niger seed medium or
alternative selective media such as sunower seed agar,
caeic acid agar and L-DOPA medium (Staib, 1962; Shaw
and Kapica, 1972; Paliwal and Randhawa, 1978; Hopfer
and Blank, 1975). Colonies of C. gattii and C. neoformans
can be presumptively identied on these media by
their characteristic, variably chocolate brown pigment.
However, other yeasts, such as melanin positive (Mel+)
variants of Cryptococcus laurentii and Cryptococcus cassiae
may also develop the same pigment (Figure 1A and1B) and
thus likely to be confused with C. gattii and C. neoformans
during their isolations in primary cultures.
Mouse-inoculation technique
e supernatant is injected intraperitoneally into labo-
ratory mice which are sacriced after 4–6 weeks. eir
visceral organs such as liver and spleen are macerated
and inoculated on appropriate mycological media for
isolation of C. neoformans and C. gattii or other target
pathogenic fungi. is method has become obsolete
for the isolation of C. neoformans/C. gattii from envi-
ronmental sources since the introduction of Staib’s
birdseed/niger ((Guizotia abyssinica) agar or other
selective media.
Phenotypic and molecular identification
Cryptococcus gattii diers from C. neoformans in various
aspects, including a contrasting human host prole and
a reduced susceptibility to certain antifungal drugs (Lin
and Heitman, 2006; Khan et al., 2007; Hagen et al., 2010;
Chowdhary et al., 2011b). Since C. gattii is as an emerg-
ing pathogen, it is important for the clinical microbiol-
ogy laboratory to dierentiate it from its closely related
C. neoformans. A prociency testing survey administered
by the New York State Department of Health indicated
that only 5% of clinical laboratories participating in the
event were able to correctly identify C. gattii, while the
remaining 95% of laboratories surveyed misidentied the
isolate as C. neoformans (New York State Department of
Figure 1. A: Chocolate brown colonies of a melanin positive
(Mel+) variant of Cryptococcus laurentii (see arrows) isolated
from decayed wood as seen in primary cuture on a niger seed agar
plate after 5 days of incubation at 28°C (After Mussa et al., 2000).
B: Chocolate brown colonies of Cryptococcus cassiae sp. nov. (see
arrows) as seen in primary culture on a niger seed plate inoculated
with decayed wood of an Acacia tree sampled from Bharatpur, UP,
India.
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4 A. Chowdhary et al.
Critical Reviews in Microbiology
Health, 2005). In comparison, the situation in developing
countries is expected to be far more unsatisfactory.
Various phenotypic techniques have been used for
dierentiating C. gattii from C. neoformans, includ-
ing use of canavanine-glycine-bromothymol blue
agar (CGB), glycine-cycloheximide-phenol red agar,
creatinine-dextrose bromothymol blue thymine agar
and creatinine-dextrose bromothymol blue agar
(Kwon-Chung et al., 1978, 1982; Salkin et al., 1982;
Min and Kwon-Chung, 1986; Irokanulo et al., 1994).
e CGB agar was reported to give fewer false-positive
and false-negative results than the others. Currently
available commercial methods for yeast identication,
such as API 20 AUX (bioMerieux, Paris, France), Vitek
(bioMerieux), and MicroScan (Siemens, Erlangen,
Germany) do not dierentiate between C. neoformans
and C. gattii. However, recently, matrix assisted laser
desorption/ionization time of ight mass spectrom-
etry (MALDI-TOF MS) has been successfully used to
dierentiate C. neoformans from C. gattii (McTaggart
et al., 2011a). Serotyping is useful for dierentiation of
C. gattii from C. neoformans but the only commercial
kit previously available for serotyping (Crypto-Check
kit; Iatron Inc., Tokyo, Japan) is no longer manufac-
tured. However, multiplex PCR and liquid array-based
methods have been recently reported for dierentia-
tion of C. neoformans and C. gattii (Bovers et al., 2007;
Feng et al., 2008), but this technology is not yet rou-
tinely utilized by most clinical laboratories. McTaggart
et al. (2011b) evaluated an algorithm, incorporating
commercial rapid biochemical tests, dierential media
and DNA sequence analysis for a rapid and accurate
dierentiation of C. gattii and C. neoformans. CGB agar
or IGS sequencing dierentiated these isolates within
48 h. On CGB, 25 of 27 (93%) C. gattii strains induced
a blue color change in contrast to 0 of 86 C. neofor-
mans isolates. Neighbour-joining cluster analysis of
IGS sequences dierentiated C. neoformans var. grubii,
C. neoformans var. neoformans and C. gattii.
Over the past two decades, a variety of molecular
techniques have been introduced for identication of
pathogenic fungi. Many of these techniques, as opposed
to classical phenotypic characterization, have the poten-
tial to provide rapid, sensitive, and specic identication
of the C. neoformans species complex. Molecular nger-
printing techniques, e.g. random amplied polymorphic
DNA (Boekhout and van Belkum, 1997), restriction
fragment length polymorphism (RFLP) (Meyer et al.,
2003; Kidd et al., 2004), pulsed-eld gel electrophoresis
(Boekhout et al., 1997), luminex technology (Bovers et al.,
2007; Diaz and Fell, 2005) amplied fragment length
polymorphism (AFLP) (Boekhout et al., 2001; Hagen
et al., 2010), PCR ngerprinting with minisatellite (M13)
or microsatellite primers (GACA4 or CTG5) (Meyer et al.,
1999; Meyer et al., 2003; Kidd et al., 2004), karyotypes
(Boekhout et al., 1997), sequencing (Diaz et al., 2000;
Katsu et al., 2004; Bovers et al., 2008; McTaggart et al.,
2011b), multilocus microsatellite typing (Illnait-Zaragozí
et al., 2010a,b), mating type locus (Cogliati et al., 2006)
and multilocus sequence typing (MLST) (Litvintseva
et al., 2006; Hiremath et al., 2008; Meyer et al., 2009;
Chowdhary et al., 2011a) are techniques that have been
applied to characterize the genetic heterogeneity of the
C. neoformans species complex. Based on molecular
studies, using PCR ngerprinting, AFLP analysis, analy-
sis of the orotidine monophosphate pyrophosphorylase
(URA5) and phospholipase (PLB1) genes by RFLP and
MLST, C. neoformans and C. gattii have been further
classied into several distinct genotypes: AFLP1/VNI and
AFLP1A/AFLP1B/VNII (C. neoformans var. grubii, sero-
type A), AFLP2/VNIV (C. neoformans var. neoformans,
serotype D), AFLP3/VNIII (hybrid serotype AD), AFLP4/
VGI, AFLP6A/VGIIa, AFLP6B/VGIIb, AFLP6C/ VGIIc,
AFLP5/VGIII, AFLP7/VGIV and AFLP10/VGIV (C. gattii,
serotype B/C). In addition, hybrids of C. neoformans var.
neoformans and C. gattii and of C. neoformans var. gru-
bii and C. gattii belong to genotypes AFLP8 and AFLP9,
respectively (Bovers et al., 2006, 2008). Although more
labour intensive and costly, DNA sequencing is rapidly
becoming a common procedure in most clinical labora-
tories because of its greater discriminatory power than is
generally provided by using dierential media and bio-
chemical tests. In laboratories where DNA sequencing is
routinely available, IGS, internal transcribed spacer (ITS)
and D2 region of the fungal 28S large ribosomal subunit
distinguish C. neoformans from C. gattii and are opti-
mal methods for identication of Cryptococcus species.
However, sequencing of the D2 region of the 28S large
ribosomal subunit may not be able to reliably distinguish
C. neoformans var. grubii and C. neoformans var. neofor-
mans (Klein et al., 2009). Similarly, sequencing of the ITS
region dierentiates C. gattii but has poor discrimination
(≥99.5% similarity) between C. neoformans var. neofor-
mans and C. neoformans var. grubii (McTaggart et al.,
2011b).
Environmental prevalence
e success in demonstration of C. neoformans in soil
and avian excreta in early studies by the stalwarts of
Medical Mycology, Chester Emmons (1951), Libero
Ajello (1958) and Maxwell Littman (1959) was achieved
by employing the mouse-inoculation technique.
Investigations on the natural habitat of C. neoformans
were further stimulated when Fritz Staib (1962) devel-
oped and introduced the niger seed agar, a selective
medium for its rapid isolation and presumptive iden-
tication. In India, the Mycology Group of Vallabhbhai
Patel Chest Institute (VPCI), Delhi, has been actively
engaged for long in studies on the environmental prev-
alence of C. neoformans and C. gattii.
Avian and other excreta
e rst environmental isolation of C. neoformans
in India was made from old pigeon excreta in Delhi
(Sethi et al., 1966), employing the mouse-inoculation
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Environmental prevalence of C. neoformans and C. gattii in India 5
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technique. e positive samples came from a heap of
dry, mortar-like excreta admixed with dust and feathers
found accumulated inside an abandoned uppermost
oor of the Arts Faculty Building, University of Delhi,
North Campus, where feral pigeons were roosting. e
isolated C. neoformans strains were found to be patho-
genic to mice experimentally infected intra-cerebrally.
ese observations were conrmed by Padhye and
irumalachar (1967) from Pune and Gugnani et al.
(1967; 1972) from Delhi who reported the isolation of
C. neoformans from old excreta collected from a pigeon
house inside the National Zoological Park, New Delhi,
and also from an old building inside the B.R. College
Agricultural Farm, Agra. Subsequently, Khan et al.
(1978) reported a more frequent association (55.4%) of
C. neoformans with old pigeon excreta in a Charity Bird
Hospital, Delhi. is was followed by a larger VPCI study,
covering 489 diverse natural substrates and employing
Staib’s niger seed agar as a selective isolation medium.
C. neoformans was reported from 38 of 253 (15%) old
avian excreta investigated (Pal et al., 1979). is report
was noteworthy for isolating C. neoformans for the rst
time from the excreta of 5 avian species, namely Ara ara-
rauna (Blue-and-yellow Macaw), Ara chloroptera (Red-
and-green Macaw), Melopsittacus undulates (Common
Pet Parakeet), Centropus sinensis (Crow Pheasant) and
Estrilda amandava (Red Munia). No serotyping or vari-
etal identication of the C. neoformans isolates was done
in any of the afore-mentioned studies. Outside of India, C.
neoformans has been reported from 5/97 (5.2%) excreta
samples of swallow (Hirundo rustica) in Iran (Hedayati
et al., 2011), 24% of chicken faeces in ailand (Kuroki
et al., 2004) and from 25.5% of excreta of caged passer-
ine and psittacine birds in Brazil (Lugarini et al., 2008).
It seems pertinent to point out here that avian excreta
is primarily a natural habitat of C. neoformans, although
C. gattii, serotype B has been sporadically isolated from
this substrate (Abegg et al., 2006). Nielsen et al. (2007)
have reported that pigeon guano supported in vitro
growth of both species, and it allowed a prolic mat-
ing of C. neoformans but not of C. gattii. Consequently
pigeon guano represents a fundamental but not a real-
ized environmental niche for C. gattii.
Mussa (1997) reported isolation of C. neoformans in 4
of 181 (2.2%) bat guano samples, collected mostly from
Delhi. In another study by the VPCI Mycology Group, C.
neoformans was incidentally isolated from the intestinal
contents of one of 155 insectivorous bats belonging to
Rhinopoma hardwickei, captured from an abandoned,
dark and dingy oor of an old school building in Delhi
(Khan et al., 1982). Outside of India, a solitary isolation of
C. gattii from bat guano has been reported from Brazil by
Lazéra et al. (1993).
Worldwide literature reports have rmly established
that desiccated excreta of pigeons and other avian spe-
cies are an excellent natural substrate for the growth and
multiplication of C. neoformans in the environment. In
saprobic settings, C. neoformans is inhibited by UV light
and temperature exceeding 44°C. It can catabolize high
concentration of urea, catecholamines and other nitrog-
enous compounds in pigeon excreta (Fiskin et al., 1990).
Also, it can produce laccase and become melanized
(Nosanchuk et al., 1999) which provides some protec-
tion against UV radiations, temperature extremes and
oxidative compounds. e melanin chelates silver and
perhaps other toxic heavy metals that protects the fun-
gus against degrading enzymes (Rosas and Casadevall,
2001; García-Rivera and Casadevall, 2001). e ability
to produce urease, enables it to thrive on urea and other
nitrogenous compounds in the excreta. C. neoformans
may produce potentially infectious basidiospores in the
pigeon excreta. In sites with pigeon manure harbouring
C. neoformans, air sampling has demonstrated aerosols
of yeast cells and or basidiospores (Litvintseva et al.,
2011). Besides, many investigators have reported the
proximity of patients with cryptococcosis to pigeon or
other avian habitats (Currie et al., 1994; Garcia-Hermoso
et al., 1997; Nosanchuk et al., 2000). In addition, isolates of
C. neoformans recovered from patients and pigeon guano
(Franzot et al., 1997; Currie et al., 1994; Litvintseva et al.,
2005) and excreta of a pet magpie, Pica pica ( Lagrou et al.,
2005) were shown to have the same genotype, suggest-
ing zoonotic transmission of cryptococcosis. erefore,
desiccated pigeon excreta constitute a plausible source
of human cryptococcosis.
Role of pigeon as carrier/host?
Pigeons and most other birds are not hosts to C. neofor-
mans and they do not acquire cryptococcosis because
their body temperature (average 42.5°C) is too high to
allow the growth of this fungal pathogen (Emmons, 1955;
Littman and Borok, 1968). Nevertheless, a number of
cases of avian cryptococcosis caused by C. neoformans
var. grubii and C. gattii have been reported, but the tol-
erance of the etiologic isolates to high temperatures was
not tested. Also, the infection in these avian cases was
restricted to cutaneous sites or the upper respiratory
tract (Malik et al., 2003).
e isolation of C. neoformans from the feet, beak
and gastrointestinal tract of the feral pigeon (Columba
livia) has been well documented (Swinne-Desgain,
1975). In India, Sethi and Randhawa (1968) carried out
a study involving experimental feeding of feral pigeons
with a virulent strain of C. neoformans. None of the
infected birds showed any signs of cryptococcal infec-
tion although C. neoformans could be isolated from the
intestinal contents or fresh excreta in 17 of 18 infected
pigeons sacriced over a period of 3 weeks. In addition,
the pathogen was recovered from the fresh excreta and
intestinal contents of one of the pigeons necropsied on
the 36th post-infection day. From the same laboratory,
Khan et al. (1978) reported the isolation of C. neoformans
from the crops of 4 (1.3%) of 319 feral pigeons investigated
in Delhi, supporting the view that the pigeon itself is not
a reservoir of the pathogen but may serve as its mechani-
cal carrier and disseminator in the environment. In this
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6 A. Chowdhary et al.
Critical Reviews in Microbiology
context reference may be made to Cafarchia et al. (2006)
who have suggested a role for birds of prey such as Falco
tinnunculus (Kestrel) and Buteo buteo (Buzzard) as carri-
ers and spreaders of C. neoformans.
Soil
Cryptococcus neoformans and C. gattii has been fre-
quently reported from soil and dust. However, these
isolations are from samples that contained excreta of
pigeons, other avian species or bats (Emmons, 1955;
Ajello, 1958; Casadeval and Perfect, 1998). In a survey
of soil-inhabiting human pathogenic fungi in India,
Gugnani and Shrivastav (1972) reported the isolation
of C. neoformans from four out of 308 soil samples,
employing the mouse-inoculation technique. ree of
the 4 positive soil samples were rich in bat guano and
collected from a historical monument, whereas the
remaining positive sample contained traces of decom-
posed plant material and originated from a riverine
site. Recently, Randhawa et al. (2008) have reported
isolation of both C. neoformans and C. gattii from
soil surrounding the base of a number of host trees
harboring these pathogens in decayed wood of their
trunk hollows. ese host trees belonged to Syzygium
cumini, Mimusops elengi, Polyalthia longifolia and
Azadirachta indica. Of the 95 soil samples investigated,
25 were positive for C. neoformans, 23 for C. gattii and
5 for both of the species, yielding a prevalence of 26%,
24% and 5%, respectively. Depending upon the site of
investigation, the prevalence in soil ranged from 11 to
50% for C. neoformans, 14–57% for C. gattii and 7–11%
for concomitant occurrence of both the species in the
same soil sample. Concerning the prevalence in soil
with regard to individual host tree species, the high-
est for C. gattii at 29% was in the vicinity of S. cumini
trees as against 25% and 20% in the vicinity of M. elengi
and A. indica trees, respectively. For C. neoformans,
the highest prevalence in soil was 31% in the vicinity
of S. cumini, followed by 12.5% each for M. elengi and
P. longifolia trees. None of the 10 control soil samples
from an open playground away from the S. cumini
trees harbouring C. neoformans yielded any isolation
of C. gattii or C. neoformans. ese ndings are in
concordance with the results of an extensive study on
characterization of environmental sources of C. gattii
including soil samples collected from within one meter
of the base of many host trees investigated in British
Columbia, Canada, and the Pacic Northwest of the US
(Kidd et al., 2007b).
Trees, decayed wood, other plant debris
Cryptococcus gattii was reported for the rst time
from environmental sources in India by Chakrabarti
et al. (1997) who isolated it from ve of 354 (1.4%)
plant debris samples of Eucalyptus trees investigated
in Punjab. e positive samples belonged to three
Eucalyptus camaldulensis trees, two of which were in
the Chak Sarkar Forest and one in the village Periana
near Ferozepur, Punjab. No quantitative results were
reported such as the pathogen’s population density in
any positive sample and no attempts were reported to
re-isolate the fungus from the positive E. camaldulen-
sis trees. Consequently, epidemiologic signicance of
their ndings remained uncertain. Gugnani et al. (2005)
have reported a low prevalence of 0.4% for C. gattii in
owers of E. terreticornis trees in Delhi and of C. neo-
formans var. grubii in the bark of E. camaldulensis trees
in Chandigarh, Punjab. In common with the preceding
report of Chakrabarti et al. (1997), no information was
provided on the population density of C. gattii and C.
neoformans in the positive samples.
In an environmental study carried out in Vellore, South
India, C. gattii was not found in any of the 86 E. camaldu-
lensis trees sampled (Abraham et al., 1997). is was in
agreement with the negative results for C. gattii reported
by Swinne et al. (1994) who investigated 657 Eucalyptus
samples collected in Rwanda, Africa, Hamasha et al.
(2004) from Jordan and Ergin et al. (2004) from Turkey
who investigated 500 and 1175 plant debris samples
related to Eucalyptus trees, respectively. Likewise, in an
investigation of 732 environmental samples in Vancouver
Island, Canada, C. gattii has not been found in any of the
Eucalyptus debris samples although it was isolated from
several native tree species such as alder (Alnus spp.),
cedar (Cedrus spp.), Douglas r (Pseudotsuga menziesii),
Garry oak (Quercus garryana) and grand r (Abies gran-
dis) (Kidd et al., 2004; Kidd et al., 2007b). Negative results
for C. gattii were also reported by Randhawa et al. (2001)
in an environmental study covering 702 samples of
diverse plant material which included 498 bark samples
(E. tereticornis - 104, E. camaldulensis - 98, unidentied
Eucalyptus species - 188) collected from Delhi, Dehradun
(Uttar Pradesh) and Amritsar (Punjab) in north-western
India. However, four isolates of C. neoformans var. grubii
and a number of other yeast-like fungi were isolated. Two
of the C. neoformans isolates came from wood debris
in tree trunk hollows of Butea monosperma, ‘the forest
ame’ and one from a trunk hollow of a Tamarindus
indica tree in New Delhi sampled during May/June 1996.
e fourth C. neoformans var. grubii isolate came from the
bark of an Eucalyptus tree in Amritsar. ese isolations
provided the rst evidence in India of decayed wood in
trunk hollows of living trees as a potential ecologic niche
other than avian excreta for C. neoformans var. grubii.
Among the additional Cryptococcus species isolated in
this study were C. laurentii - 3 isolates and C. albidus -
2 isolates. Of these, 2 isolates of C. laurentii and one of
C. albidus originated from the bark of a Eucalyptus tree.
Interestingly, the third C. laurentii isolate was a rarely
reported, melanin forming (Mel+) variant that was ini-
tially mistaken for C. neoformans. It was repeatedly iso-
lated from wood debris inside hollows of an abandoned,
old timber beam of sal wood, Shorea robusta, sampled
serially during September 3, 1993 to March 12, 1996. As
the variant isolate possessed phenoloxidase (melanin
forming) activity and it caused lesions in liver and spleen
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Environmental prevalence of C. neoformans and C. gattii in India 7
© 2012 Informa Healthcare USA, Inc.
in experimentally infected laboratory mice, the ndings
are of potential clinical and epidemiologic signicance
(Mussa et al., 2000).
e afore-mentioned study led to more intensive
investigations that aimed to characterize the environ-
mental reservoirs of C. gattii and C. neoformans. It was
found that F. religiosa trees harbored only C. neoformans
var. grubii in their trunk hollows, whereas this variety and
C. gattii, serotype B, were both equally distributed (prev-
alence 10.6%) in decayed wood inside trunk hollows of
the 66 S. cumini trees investigated. Furthermore, C. gattii
was repeatedly isolated on 36/44 (82%) occasions from
7 S. cumini known positive trees sampled longitudinally
over a period of 689 days. Likewise, the overall isolation
frequency of C. neoformans var. grubii from the two host
tree species came to 22/27 (81%) occasions during the
same follow-up span. ese data strongly supported a
long-term colonization of decayed wood inside trunk
hollows of S. cumini by both the pathogens. e conclu-
sion was reinforced by high population densities found
in wood samples (maximally 6 × 105 cfu/g for C. gattii and
8 × 104 cfu/g for C. neoformans). No Eucalyptus trees were
seen in the localities where S. cumini and F. religiosa host
trees were sampled. During the next phase of the study
(Randhawa et al., 2006), a large number of S. cumini
trees were investigated from Delhi and other parts of
north-western India, i.e. Amritsar (Punjab), Meerut and
Bulandshahr (Uttar Pradesh) and the Chandigarh Union
Territory. e results corroborated the strong ecological
association of C. gattii and C. neoformans with S. cumini
trees throughout north-western India.
Host tree species spectrum
An overview of the environmental studies done in India
(Table 1) indicates that 17 species of trees representing 12
families have been documented as hosts of the C. neofor-
mans species complex. A decade-long study (2001–2011)
of Randhawa and coworkers, revealed that the preva-
lence of C. gattii and C. neoformans diered considerably
not only from one host tree to another but also among
trees of the same host species occurring in a given local-
ity or in dierent geographic regions. e isolation of
C. neoformans var. grubii from 17 host tree species and
of C. gattii serotype B from 12 tree species showed that
the former has a more widespread arboreal distribu-
tion. Of the C. gattii host trees, S. cumini in Delhi yielded
Table 1. Prevalence of Cryptococcus neoformans and C. gattii in decayed wood, bark or other plant debris of host trees reported
from India.
Host trees (Family)
No. samples
examined
Prevalence (%) Serotype, mating
type, genotype ReferencesC. gattii C. neoformans
Eucalyptus camaldulensis (Myrtaceae) 354 1.4 0 B Chakrabarti et al., 1997
E. camaldulensis and E. tereticornis
(Myrtaceae)
86 0 0 – Abraham et al., 1997
E. camuldulensis and Eucalyptus sp.
(Myrtaceae)
390 0 0.2 A, MATα, VNI Randhawa et al., 2001
E. camaldulensis and E. tereticornis
(Myrtaceae)
233 0.4 0.4 B, A MATα,Gugnani et al., 2005
Eucalyptus sp. 19 5.2*0B, MATα, VGI Randhawa et al., 2008
Eucalyptus globulus (Myrtaceae) 11 0 9 – Girish et al., 2011
Butea monosperma (Papilionaceae) 5 0 40 A Randhawa et al., 2001
Tamarindus indica (Papilionaceae) 2 0 50 A Randhawa et al., 2001
66 10.6 10.6 B, A, MATα, VGI,VNI Randhawa et al., 2003
Syzygium cumini (Myrtaceae) 67 9–89 0–54 B, A, MATα, VGI, VNI Randhawa et al., 2006
20 10*0B, MATα, VGI, Randhawa et al., 2008
Polyalthia longifolia (Annonaceae) 55 0–16.6 0–33.3 B, A, MATα, VGI, VNI Randhawa et al., 2008
5 20 0 B Girish et al., 2011
Mangifera indica (Anacardiaceae) 38 0 0–14.2 A, MATα, VNI Randhawa et al., 2008
Azadirachta indica (Meliaceae) 35 35–40 40–80 B, A, MATα, VGI, VNI Randhawa et al., 2008
Cassia stula (Caesalpinioideae) 19 5.2 47 B, A, MATα, VGI, VNI Randhawa et al., 2008
C. marginata (Caesalpinioideae) 5 20 B Girish et al., 2011
Acacia nilotica (Mimosoideae) 19 5.2 0–21 B, A, MATα, VGI, VNI Randhawa et al., 2008
Alstonia scholaris (Apocynaceae) 19 0 5.2*A, MATα, VNI Randhawa et al., 2008
Ficus religiosa (Moraceae) 31†0 0 – Randhawa et al., 2001
17 0 17.6*A, MATα, VNI Randhawa et al., 2003
15 0 6.6 A, MATα, VNI Randhawa et al., 2008
Mimusops elengi (Sapotaceae) 13 30.7 15.3 B, A, MATα, VGI, VNI Randhawa et al., 2008
Dalbergia sissoo (Faboideae) 13 0 7.6*A, MATα, VNI Randhawa et al., 2008
Manilkara hexandra (Sapotaceae) 6 33.3 50 B, A, MATα, VGI, VNI Randhawa et al., 2008
Aegle marmelos (Rutaceae) 1 0 +#A, MATα, VNI Randhawa et al., 2008
*Attempts at re-isolation were negative; †All of these were bark samples; #Percentage not given because only a solitary sample was tested.
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8 A. Chowdhary et al.
Critical Reviews in Microbiology
the highest prevalence (89%), followed by A. indica
(35–40%), Manilkara hexandra (33%), M. elengi (31%),
Cassia marginata (20%), P. longifolia (7%), Cassia stula
and Acacia nilotica (5.2% each). Notably, the isolation
frequency of C. gattii and C. neoformans from their host
tree species was not related to the phenological state of
the trees, which was in agreement with the observations
of Lazéra et al. (1998), Granados and Castaňeda (2005),
Kidd et al. (2007b) and Byrnes et al. (2011). Furthermore,
Eucalyptus species proved to be among the least impor-
tant host for C. gattii. Both of these observations were in
striking contrast to the results reported from Australia
(Ellis and Pfeier, 1990a) where C. gattii isolations were
linked to owering of the trees, and Eucalyptus species
were reported as predominant, if not virtually exclusive
hosts. However, it is not clear as to what extent this dier-
ence can be attributed to the fact that tree species other
than those of Eucalyptus have received scant attention of
the Australian investigators interested in the ecology of
C. gattii and C. neoformans.
For C. neoformans, the most important host tree was
A. indica (prevalence 60%), followed by S. cumini (54%),
M. hexandra (50%), C. stula (47%), M. elengi, (15%),
P. longifolia, (13%) and lower prevalence in the range of
5 - 8% for Alstonia scholaris, F. religiosa and Dalbergia sis-
soo. Most of the afore-mentioned host tree species had
large canopies and conspicuous trunk hollows. As with
C. gattii, Eucalyptus trees were among the least important
host trees for C. neoformans. It seems pertinent to men-
tion here that a much larger number of host tree species
other than those recorded in India have been reported
from other countries. is includes more than 10 tree spe-
cies each from Colombia and Vancouver Island, Canada
(Granados and Castaňeda, 2005; Kidd et al., 2007b).
Interestingly, C. gattii has also been frequently isolated
from a succulent cactus species Cephalpcereus royenii in
the Guanica Dry Forest, Puerto Rico (Loperena-Alvarez
et al., 2010). It is anticipated that the current global list of
host tree species for the two pathogens (already exceed-
ing 50) will expand considerably with further ecologic
studies in as yet unexplored and climatically divergent
geographic regions in India or elsewhere.
Cryptococcus gattii, serotype C, has not yet been
reported from environmental or clinical sources in India.
Much of the available information about this pathogen has
been contributed by Elizabeth Casteñeda and coworkers
from Colombia where it occurs in association with tropical
almond trees (Terminalia catappa). In an experimental
study aimed at exploring the interaction between serotype
C and Terminalia catappa, they inoculated stems of 30
seedlings with an almond isolate. e isolate caused no
visible lesions or microscopic phytopathology, but it was
recovered in culture up to one year and also demonstrated
microscopically in sections of infected stem. It was further
observed that the fungus spread from infected stem to
soil and back to the seedlings, which was indicative of the
anity of serotype C for almond plants and of the potential
for long- term establishment of a stable inter-relationship
(Callejas et al., 1998; Escandón et al., 2002). ese observa-
tions suggest the probability of an endophytic relationship
between C. gattii and its host plant T. catappa.
Longitudinal surveillance and population
density
Of all the host trees harboring C. gattii and C. neofor-
mans identied in India, it is S. cumini that has been
most extensively investigated and documented regard-
ing its epidemiologic importance. In order to determine
whether this tree species was transiently, intermittently
or perennially colonized, 7 of the known positive S.
cumini trees in Delhi were subjected to a long-term
mycological surveillance. Signicantly, C. gattii and C.
neoformans were repeatedly re-isolated over a follow-up
period of 4.2–5.2 years. Furthermore, the positive sam-
ples carried a population density, ranging from 3 × 103 to
6 × 105 CFU/g for C. gattii and 2 × 103− 8 × 104 CFU/g for C.
neoformans (Randhawa et al., 2006). e highest popula-
tion density of C. gattii previously reported in any wood
debris sample was 2.6 × 103 CFU/g in a Cassia grandis
tree in Brazil (Lazéra et al., 2000) which is 1/230th part of
the peak density of 6 × 105 CFU/g in one of the S. cumini
wood samples. It would have been interesting to compare
the population density data of Randhawa et al. (2006)
with those of the Eucalyptus debris samples reported
positive for C. gattii from Australia by Ellis and Pfeier
(1990a) but, unfortunately, no such data are available.
From a review of the environmental studies done so far
in India and elsewhere, it is clear that the natural habitat
of C. gattii is not restricted to plant debris of Eucalyptus
species or any other specic tree species. Instead, it has
a generalized ecologic association with decayed wood
or other plant debris of a wide spectrum of diverse tree
species. e major factor underlying wood colonization
by C. gattii and C. neoformans is probably the ability of
these pathogens to produce the enzyme laccase which
has been implicated in degradation of wood lignin by
Basidiomycetes (Kirk and Farrel, 1987; urson, 1994;
Williamson, 1994; Eggert et al., 1996).
Australian hypothesis of global
spread of C. gattii
In their historically important papers on the ecology of
C. gattii, Ellis and Pfeier (1990a,b) and Pfeier and Ellis
(1992) reported that owers and other plant debris of E.
camaldulensis and E. tereticornis trees constituted the
main natural habitat of C. gattii. ey also hypothesized
that C. gattii had spread globally to other countries
through the Australian export of infected E. camaldu-
lensis seeds containing dormant dikaryotic mycelium
of C. gattii. ey believed that C. gattii was a smut-like
fungus which, however, belongs to an entirely unrelated
group of plant pathogens classied under the class
Ustilaginomycetes, subphylum Ustilaginomycotina,
whereas C. gattii belongs to the class Tremellomycetes,
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Environmental prevalence of C. neoformans and C. gattii in India 9
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subphylum Agaricomycotina. In this probably misplaced
analogy with a plant pathogen, it was further speculated
that when the infected seeds of E. camaldulensis would
germinate and grow, the mycelia of C. gattii would also
grow in the tissues of the seedling and eventually sporu-
late to produce basidiospores at the time of the host tree’s
owering (Ellis and Pfeier, 1990b). e authors stressed
this hypothesis in various publications despite lack of any
supportive scientic evidence and it continues to be cited
(Litvintseva et al., 2011). e weakness of this hypothesis
is apparent from the following observations: One, C. gattii
has never been isolated from seeds, ovaries or anthers of
E. camaldulensis or any other Eucalyptus species, nor has
any histological evidence been presented to demonstrate
the presence of dikaryotic mycelium in any of these or
other parts of the host tree. Incidentally, attempts, in our
laboratory to demonstrate the fungus histologically in
situ in decayed wood pieces taken from inside trunk hol-
lows of S. cumini positive trees in Delhi were unsuccess-
ful (Randhawa and Kowshik, unpublished data). Two,
the smut fungi are plant pathogens, whereas C. gattii is
essentially an environmental, free-living saprobe which
may sporadically infect humans and animals. ree, C.
gattii has been reported from native tree species in many
countries, including those which have no Eucalyptus
ora. To cite two illustrative examples, C. gattii has been
isolated from a native jungle tree, Guettarda acreana, in
a wild tropical forest without anthropic action in Brazil
(Fortes et al., 2001) and also from a Douglas r tree
(Pseudotsuga menziesii) in Berg en Dal, Nijmegen, the
Netherlands, a region where no tropical Eucalyptus trees
are prevalent (Chowdhary and Meis, unpublished data).
Last but not the least, the hypothesis is conceptually
wrong because it was based on the presumption that C.
gattii was a constituent of mycobiota, exclusively native
to Australia and that the fungus had no autochthonous
occurrence in other parts of the world.
Fruits and vegetables
Fruits and vegetables do not constitute a natural habi-
tat or ecological niche for C. neoformans and C. gat-
tii although these pathogens have been sporadically
isolated from these substrates. e rst report on the
occurrence of C. neoformans in fruits was by Staib et al.
(1972; 1973) from Germany who isolated it from sliced,
ripe peach fruits incubated in the laboratory. In another
report (López-Martínez and Castañón-Olivares, 1995),
C. neoformans var. neoformans was isolated from 9.4%
of fruits and 4.2% of vegetables in Mexico City. In India,
C. neoformans has been reported from solitary samples
of tomato (Lycopersicon esculentum), ‘vegetable sponge’
(Lua cylindrica) and brinjal (Solanum melongena) in
a survey of 437 samples of a wide variety of vegetables
collected from a number of markets in Delhi. Serotyping
revealed that 2 of the 3 isolates were serotype A whereas
one was untypeable (Misra, 1978; Misra and Randhawa,
2000). In addition, another study from Delhi has reported
isolation of C. neoformans from solitary samples of a
few additional vegetables and fruits in an investigation
covering 254 vegetables and 186 fruits samples (Pal and
Mehrotra, 1985). Most likely, the positive vegetables and
fruits in the afore-mentioned reports carried the patho-
gen as an environmental contaminant.
Water, other substrates
e occurrence of C. gattii and C. neoformans in water
remains as yet unexplored in India. Interestingly, a highly
virulent genotype of C. gattii, VGII, has been frequently
isolated from fresh water as well as marine water in
British Colombia, Canada, which is an endemic area
for this pathogen (Kidd et al., 2007b). e prevalence
rate and population density of the new genotype var-
ied from 16.6% (1.1 ± 12 CFU/100ml) to 20.7% (5.1 ± 11
CFU/100 ml) in fresh water of lakes and rivers/ creeks
whereas it was 21% (2.2 ± 2.0 CFU/100 ml) in seawater.
Kidd et al. (2007b) further demonstrated that this C. gat-
tii genotype survived best in ltered or unltered ocean
water at room temperature and distilled water at room
temperature.
Concerning other environmental isolations, there is
a solitary report on the isolation of C. neoformans var.
grubii from one of the 15 beehives examined from the
Karabucak Eucalyptus forest in Tarsus, Turkey (Ergin
et al., 2004). In Uruguay, C. gattii has been reported from
the nest of a wasp, Polybia occidentalis (Gezuele et al.,
1993). Also, there is a report on the isolation of C. gattii
from insect frass in Canada (Kidd et al., 2003). Besides,
Swinne et al. (1986) have reported C. neoformans var.
neoformans from the digestive tract of cockroaches in
Kinshasa, Democratic Republic of the Congo in Africa.
However, the role of cockroaches and other insects as
vectors in the spread of C. neoformans in the environ-
ment is unknown.
Aerial survey
Information on the aerial prevalence of C. neoformans
and C. gattii in India is scarce. e rst report is by Khan
et al. (1978) who isolated C. neoformans by aerial expo-
sure of Staib’s niger seed plates in a Charity Bird Hospital,
Delhi, which had over 600 pigeons. It was observed that
the isolation frequency of C. neoformans was much
higher on petri-dishes exposed inside the pigeon cages
(55%) than in those exposed outside the cages in the hos-
pital room (30%). Notably, the fungus could no longer
be isolated from the indoor air after a thorough cleaning
and painting of the entire bird hospital. Randhawa and
Paliwal (1979) reported negative results for C. neofor-
mans in a 2-year, aeromycological study (1975–1977)
conducted outdoor in the lawns of a postgraduate hostel
in the University of Delhi, North Campus. However, other
species of Cryptococcus, such as, C. ater, C. avus, C. lau-
rentii, C. magnus, C. terreus, C. uniguttulatus and C. albi-
dus were occasionally isolated. Recently, isolations of the
C. neoformans species complex were frequently made by
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10 A. Chowdhary et al.
Critical Reviews in Microbiology
sampling of air inside tree trunk hollows of S. cumini trees
in Delhi which were known to harbor C. neoformans and
C. gattii (Randhawa et al., 2006). Attention may be called
here to the aerial prevalence of C. gattii in Vancouver
Island, Canada. e reported C. gattii concentrations
(CFU/m3) in air samples were found to be signicantly
higher during the warm, dry summer months, although
potentially infectious propagules (less than 3.3 µm in
diameter) were present throughout the year (Kidd et al.,
2007b).
Seasonal variations
It is understood that abiotic factors such as pH, humid-
ity, temperature, sunlight and wind play an important
role in the environmental prevalence and dissemination
of C. neoformans and C. gattii. Most of the work done so
far is on the occurrence of C. neoformans var. grubii in
avian excreta which support its growth and multiplica-
tion under dry conditions (Ruiz et al., 1981; Caicedo
et al., 1999; Montenegro and Paula, 2000; Kuroki et al.,
2004; Grandos and Castañeda, 2005). In vitro stud-
ies by Martinez et al. (2001) have shown dierences in
thermo-tolerance between C. neoformans var. grubii
(serotypes A) and C. neoformans var. neoformans (sero-
type D). However, the problem of seasonal variations in
environmental prevalence of C. neoformans and C. gat-
tii remains virtually unexplored. Recently, Randhawa
et al. (2011) have reported a retrospective study of sea-
sonal variations in the prevalence of these pathogens in
decayed wood inside trunk hollows of a wide spectrum
of tree species investigated from ve geographical loca-
tions in north-western India over a period of 7 years
(2000–2007). Climatically, north-western India has ve
distinct seasons, namely winter, spring, summer, rainy
season and autumn. e data analyzed included results
of isolation of C. neoformans and C. gattii from 1439
decayed wood samples collected from trunk hollows of
518 trees, representing 20 species. Of the 406 isolates of
C. neoformans species complex, 247 were C. neoformans
var. grubii (serotype A) and 171 were C. gattii, serotype B.
Both pathogens were isolated during all the seasons, and
the overall prevalence of C. neoformans var. grubii was
signicantly higher (17.2%) than that of C. gattii serotype
B (11.9%, p < 0.0001). It indicated that decayed wood was
as good, if not a better natural habitat for C. neoformans
var. grubii as for C. gattii. Both of the pathogens revealed
some seasonal variations in their prevalence, the highest
being during the autumn, followed by that in the summer.
For C. gattii, the prevalence during the winter was signi-
cantly less than that during the summer (p < 0.02) and the
autumn (p < 0.02). In contrast, the lowest prevalence of
C. neoformans var. grubii (10.7%) was in the rainy sea-
son which was signicantly less than that in the autumn
(p < 0.0001), followed by that in the summer (p < 0.0001)
and winter (p < 0.001). Interestingly, a similar pattern of
low prevalence of C. neoformans var grubii in chicken
faeces during rainy season and high prevalence during
the dry season has been reported from ailand (Kuroki
et al., 2004). On the other hand, the low prevalence of C.
gattii in decayed wood during winter was similar to that
reported from Bogotá, Colombia, where C. gattii had a
low population density in bark samples but it was not
found in decayed wood of trunk hollows investigated
during January and February (Granados and Castañeda,
2005). Comprehensive prospective studies are warranted
in order to gain an insightful knowledge of any seasonal
pattern of prevalence of C. neoformans and C. gattii not
only in decayed wood but also in other natural substrates
such as avian excreta and soil.
Population structure
Population structure denotes genetic diversity among
individuals constituting a population, their operative
modes of reproduction, genetic exchange and formation
of subgroups which may be determined by geographical,
temporal and other ecological factors. Much of the avail-
able information on the environmental population struc-
ture of C. neoformans and C. gattii in India has resulted
from a collaborative study between the Mycology group
of VPCI and Dr. Jianping Xu, Department of Biology,
McMaster University, Hamilton, Canada. e main nd-
ings of this study are summarized below:
C. neoformans
e rst paper reported on the structure of environmen-
tal populations of C. neoformans var. grubii, compris-
ing 78 isolates originating from decayed wood in trunk
hollows of 9 tree species in 5 geographical locations, i.e.
Union Territory of Delhi, Bulandshahr and Hathras (Uttar
Pradesh), Amritsar (Punjab) and Amrouli (Haryana) in
north-western India (Hiremath et al., 2008). e isolates
were subjected to MLST, using ve gene fragments. All
of the isolates were found to be molecular type VNI and
mating type α (MATα). Population-genetic analyses pro-
vided no evidence for signicant dierentiation among
populations belonging to either dierent geographical
areas or dierent host tree species. Interestingly, despite
the lack of mating type a (MATa) strains, unambiguous
evidence for recombination was observed which sup-
ported the hypothesis that strains of C. neoformans may
undergo sexual reproduction on decaying wood of vari-
ous host tree species.
C. gattii
e second paper in the series dealt with genotyping of
109 isolates of C. gattii, serotype B, originating from the
wood detritus of trees and the surrounding soil from nine
dierent tree species at seven north-western locations,
i.e. Amritsar, Union Territories of Chandigarh and Delhi,
Amrouli, Bulandshahr, Hathras and Meerut and one in
Tamil Nadu, namely, Tiruvannamalai in south India
(Chowdhary et al., 2011a). MLST, using nine gene frag-
ments, revealed that all of the C. gattii isolates belonged
to the mating type, MATα which conforms to the global
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Environmental prevalence of C. neoformans and C. gattii in India 11
© 2012 Informa Healthcare USA, Inc.
mating type pattern of C. neoformans and C. gattii.
Molecular phylogenetic analyses identied that all of the
109 strains analyzed belonged to the AFLP4/VGI lineage
which has a wider geographic distribution than lineages
AFLP6/VGII, AFLP5/VGIII and AFLP7/VGIV (Xu, 2010;
Litvintseva et al., 2011). However, the wide distribution
of AFLP4/VGI in India has not obscured the genetic dif-
ferentiation among populations from either dierent
geographic areas or dierent host tree species in India.
Population-genetic analyses revealed limited evidence
of recombination but unambiguous evidence for clonal
reproduction and expansion.
Overall, the serotype distribution of clinical isolates so
far reported from India corresponds to the environmen-
tal distribution pattern with predominance of serotype A
strains, and a low prevalence of B, C, D, and AD (Banerjee
et al., 2004; Cogliati et al., 2011; Padhye et al., 1993).
Recently, we reported the serotypes, genotypes and mat-
ing types of 308 isolates of C. neoformans species com-
plex, originating from clinical and environmental sources.
eir genotypes were determined based on two methods:
(i) PCR ngerprinting using (GACA)4 and M13phage core
sequences as single primers and (ii) DNA sequences at
the URA5 locus. All of the C. neoformans isolates (n = 246)
were serotype A, genotype AFLP1/VNI and mating type
α, whereas all of the C. gattii (n = 62) isolates belonged
to serotype B, genotype AFLP4/VGI and mating type α.
Of the 246 C. neoformans var. grubii isolates, 160 were
clinical, originating from 130 patients, and the remaining
86 were from decayed wood of trees and soil. Among the
62 C. gattii isolates, 60 were from environmental and two
were from clinical sources. e clinical isolates had been
collected during 2002–2009 from various hospitals in the
Union Territories of Delhi and Chandigarh, and the states
of Uttar Pradesh and Himachal Pradesh (Chowdhary
et al., 2011b). In an earlier study, Jain et al. (2005) deter-
mined the genotypes in 57 clinical isolates from India. Of
these, 51 (89.4%) belonged to group VNI (C. neoformans
var. grubii, serotype A), one belonged to group VNIV (C.
neoformans var. neoformans, serotype D) and 5 isolates
belonged to group VGII (C. gattii). Consistent with the
global pattern, 90% of the Indian serotype A and B iso-
lates exhibited a MATα mating type. Forty-eight of the 51
C. neoformans var. grubii, were MATα and 3 were MATa.
e solitary isolate of C. neoformans var. neoformans was
MATα, whereas 4 of the 5 C. gattii isolates were MATα
and one MATa.
Antifungal susceptibility profiles
Resistance to antifungal agents in environmental and
clinical strains of C. neoformans and C. gattii has been
a rare global occurrence. Soares et al. (2005) reported a
solitary isolate of C. neoformans var. grubii from pigeon
excreta that was resistant to uconazole, (MIC 64 mg/L).
Similar ndings were reported from Cuba and those
authors concluded that environmental isolates seemed
to be less susceptible to uconazole than clinical ones
(Illnait-Zaragozi et al., 2008). Likewise, in another report
from Brazil, one of the environmental isolates of C. neo-
formans var. neoformans was found to be resistant to
itraconazole whereas three additional isolates exhibited
high MICs of 16–32 mg/L against uconazole (Costa
et al., 2010). e rst antifungal susceptibility testing
report on environmental isolates from India was based
on 117 isolates of C. neoformans, serotype A, and 65 of
C. gattii, serotype B, originating from decayed wood in
trunk hollows of F. religiosa and S. cumini trees, employ-
ing the Etest method (Khan et al., 2007). A comparison of
the geometric mean MICs revealed that C. gattii was less
susceptible than C. neoformans to amphotericin B (0.075
versus 0.051, p = 0.0003), uconazole (2.912 versus 2.316,
p = 0.0003) itraconazole (0.198 versus 0.034, p < 0.0001),
ketoconazole (0.072 versus 0.037, p < 0.0001), and vori-
conazole (0.045 versus 0.023, p < 0.0001). No primary
resistance was observed against amphotericin B, u-
conazole, itraconazole, ketoconazole and voriconazole
which is in consonance with worldwide literature reports
that resistance in Cryptococcus species complex is rarely
observed (Pfaller et al., 2005). In an extension of this
work, the same investigators reported the antifungal sus-
ceptibility proles in clinical and environmental isolates
of C. neoformans var. grubii, genotype AFLP1/VNI MATα
(n = 246), and C. gattii, serotype B, genotype AFLP4/VGI,
MATα (n = 62), using the broth microdilution method
(Chowdhary et al., 2011b). Both the species had low MICs
to the antifungals tested except for two clinical C. neofor-
mans var. grubii isolates that were resistant to 5-ucyto-
sine (MIC 64 mg/L). Data on the geometric mean of MICs
revealed that C. gattii was signicantly less susceptible
than C. neoformans var. grubii to uconazole, itracon-
azole and voriconazole (p < 0.0001). In addition, the MIC90
of C. gattii was 2-fold higher than that of C. neoformans
var. grubii for uconazole, itraconazole and voricon-
azole. However, no statistically signicant dierence was
observed in susceptibility of the two Cryptococcus species
to amphotericin B and 5-ucytosine. Furthermore, the
environmental C. neoformans var. grubii isolates were
signicantly less susceptible to uconazole, itraconazole
and 5-ucytosine (p < 0.0001) than the clinical isolates.
Similar results have been reported previously from Cuba
(Illnait-Zaragozi et al., 2008). Hagen et al. (2010) reported
that C. gattii, showed lower MICs for AFLP4/VGI isolates
(1.401 and 2.467 mg/L) versus the higher MICs for AFLP6/
VGII isolates (4.961 and 5.638 mg/L) against 5-ucytosine
and uconazole, respectively. Iqbal et al. (2010) tested
43 clinical isolates of C. gattii from patients in Oregon,
USA. Interestingly, their AFLP4/VGI and AFLP5/VGIII
isolates had comparatively low uconazole MICs, whilst
the majority with high MICs of 16–32 mg /L were of sub-
type AFLP6C/VGIIc. In contrast ompson et al. (2009)
reported no dierences in the antifungal susceptibilities
of the two species.
An earlier study from India had reported only the
uconazole susceptibility proles and genotypes of 57
clinical isolates, comprising 51 C. neoformans var. grubii,
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12 A. Chowdhary et al.
Critical Reviews in Microbiology
genotype AFLP1/VNI, one C. neoformans var. neofor-
mans, genotype AFLP2/VNIV, and ve C. gattii strains,
genotype AFLP6/VGII (Jain et al., 2005). e reported
MICs ranged from 8 to 16 mg/L for C. neoformans var.
grubii and 2 to 64 mg/L for C. gattii.
Future perspective
e geoclimatically divergent regions of India with their
rich variety of ora and fauna oer a wide scope for fur-
ther investigations on the environmental prevalence of
C. gattii and C. neoformans and their population struc-
ture. Currently, the occurrence of C. gattii serotype C in
the environment in India is entirely unknown. Likewise,
there are scarcely any data concerning the environmental
distribution of C. neoformans var. neoformans (serotype
D) which is largely known from Europe. Further studies
are required to compare the genetic structure of clinical
and environmental strains of both pathogens with a view
to probing the extent of their inter-relationship. Such a
study may shed a new light on the origin of subgroups
of various genotypes. ere is little information at pres-
ent on the prevalence of cryptococcosis in animals or
humans in the areas of north-western India where host
trees are perennially colonized by C. gattii and C. neofor-
mans. Comprehensive clinico-mycological investigations
are warranted to probe the magnitude of health hazard
posed by the environmental prevalence of C. gattii and
C. neoformans.
Acknowledgements
Acknowledgment is made to the Indian National Science
Academy, New Delhi, for the award of an Honorary
Scientist position to H. S. R. is work was written during
a research fellowship of A.C at the Canisius Wilhelmina
Hospital, Nijmegen, the Netherlands.
Declaration of interest
is work was nancially supported by the Department
of Science and Technology, Government of India (F.No.
SR/SO/HS-62/2008), the Indian Council of Medical
Research, New Delhi (HIV/50/107/2008) and Labland
BV, Wijchen, e Netherlands. J.F.M. received grants
form Astellas, Merck, Basilea and Schering-Plough. He
has been a consultant to Basilea and Merck and received
speakers fees from Merck, Pzer, Schering-Plough,
Gilead and Janssen Pharmaceutica. All other authors: no
potential conicts of interest.
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