JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2004, p. 1356–1359
0095-1137/04/$08.00⫹0 DOI: 10.1128/JCM.42.3.1356–1359.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 42, No. 3
Cryptococcus neoformans Shows a Remarkable Genotypic Diversity
in Brazil
M. T. Barreto de Oliveira,1 T. Boekhout,2,3 B. Theelen,2 F. Hagen,2 F. A. Baroni,4
M. S. Lazera,5 K. B. Lengeler,6 J. Heitman,7 I. N. G. Rivera,8
and C. R. Paula8*
Received 23 April 2003/Returned for modification 8 July 2003/Accepted 9 October 2003
The genotypic diversity of Brazilian Cryptococcus neoformans strains was analyzed. The majority of the
samples were ␣A (65%), followed by ␣B (17.5%), ␣D (9%), ␣AaD hybrids (5%), and ␣C (3.5%). A considerable
genotypic diversity occurred within C. neoformans var. grubii, and a new amplified fragment length polymorphism genotype, 1B, was recognized.
Cryptococcus neoformans is frequently implicated in meningoencephalitis in immunocompromised patients (17, 18) and is
commonly associated with pigeon droppings and plant materials (1, 3, 4, 6, 7, 13, 14, 16–18, 19, 23).
Amplified fragment length polymorphism (AFLP) genotyping has been applied to evaluate the genetic structure of C.
neoformans (2, 12). In this report, we analyzed genotypes,
serotypes, and mating types of 58 Brazilian isolates of the C.
neoformans complex, including 21 isolates from patients diagnosed prior to the onset of the AIDS pandemic, 10 isolates
from AIDS patients, and 27 isolates from environmental
sources (Fig. 1). These strains belong to the Culture Collection
of the Biomedical Science Institute, University of São Paulo,
São Paulo, Brazil. Strains ICB 59 (⫽ CBS 132, serotype AD),
ICB 162 (⫽ NIH 444, serotype C) and ICB 163 (⫽ NIH 442,
serotype D) were used as reference strains.
Serotypes were determined by the Crypto Check kit (Iatron,
Tokyo, Japan) (11) and by PCR using serotype- and matingtype-specific primers (Table 1). For C. neoformans (⫽ C. neoformans var. neoformans and grubii), primers were based on
STE12 and GPA1 genes (15), as well as some newly developed
primers obtained by using STE20 gene sequences. PCR was
performed under the following conditions: primer pair
JOHE1671/1672, 25 cycles of 96°C for 30 s, 65°C for 30 s, and
72°C for 30 s; primer pairs JOHE3240/JOHE2596 and
JOHE3241/JOHE2596, 25 cycles of 96°C for 30 s, 55°C for 30 s,
and 72°C for 45 s; and primer pairs JOHE7264/JOHE7265,
JOHE7267/JOHE7268, JOHE7270/JOHE7272, and JOHE7273/
JOHE7275, 30 cycles of 96°C for 30 s, 66°C for 30 s, and 72°C
for 60 s. The mating types of the Cryptococcus gattii isolates
were determined by using primers designed by Chaturvedi et
al. (5) and Halliday et al. (9).
Using serotype-specific antibodies, 38 isolates were serotype
A, 10 were serotype B, 6 were serotype D, 2 were serotype C,
and 1 was serotype AD (Fig. 1). Serotypes of C. neoformans
(serotypes A, D, and AD) analyzed by PCR were in most cases
concordant with results obtained by the agglutination test. Isolates ICB 160 and ICB 155, however, were found to be serotype AD by the PCR-based method, while the Iatron kit gave
serotype A. These findings agree with the previously observed
serotype variation among AD hybrid strains analyzed by agglutination (10, 15). Our results showed a predominance of
serotypes A (66.7%) and B (17.5%) in the Brazilian populations studied, thus confirming earlier reports (4, 19, 20–22).
AFLP analysis, performed according to Boekhout et al. (2),
revealed two main genotypic clusters, corresponding with the
species C. neoformans (AFLP genotypes 1 to 3) and C. gattii (⫽
C. bacillisporus ⫽ C. neoformans var. gattii, AFLP genotypes 4
to 6). Thirty-seven strains were AFLP genotype 1, including 1
AFLP genotype 1A isolate (ICB 154); 5 isolates were AFLP
genotype 2; 3 were AFLP genotype 3; 2 were AFLP genotype
4; 3 were AFLP genotype 5; and 7 were AFLP genotype 6 (Fig.
1). Strains ICB 155 and 160, which were found to be serotype
A by agglutination, belonged to AFLP genotype 3, thus supporting the AD hybrid nature (2). At the genotypic level, our
results agree with recent observations published by Casali et al.
(4). However, using AFLP, we were able to demonstrate considerable genotypic divergence within the Brazilian isolates of
the C. neoformans complex, because many polymorphic AFLP
markers occurred. Therefore, our AFLP data seem to conflict
with previous genotypic data for Brazilian isolates of C. neoformans (8). In particular, isolates of C. neoformans var. grubii
(serotype A, AFLP genotype 1) showed an unexpected diversity (Fig. 1 and 2). Five serotype A MAT␣ isolates showed only
approximately 50% similarity to the remaining serotype A
* Corresponding author. Mailing address: Av. Prof. Lineu Prestes,
1374, Cidade Universitária—USP, CEP 05508-900 São Paulo, Brazil.
Phone: 551130917294. Fax: 551130917354. E-mail: crpmicol@uol.com.br.
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Department of Microbiology and Parasitology, Federal University of Rio Grande do Norte, Natal,1 Department
of Microbiology and Immunology, University Rural of Rio de Janeiro,4 and FIOCRUZ,5 Rio de
Janeiro, and Department of Microbiology, Biomedical Science Institute, University of
São Paulo, São Paulo,8 Brazil; Centraalbureau voor Schimmelcultures,2 and
Department of Medicine, University Medical Center,3 Utrecht, The Netherlands;
Department of Microbiology, Heinrich-Heine-University of Düsseldorf,
Düsseldorf, Germany6; and Duke University,
Durham, North Carolina 277087
VOL. 42, 2004
NOTES
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FIG. 1. Clustering of AFLP banding patterns of Brazilian isolates of C. neoformans and C. gattii (C. bacillisporus). (ICB 102 was demonstrated
to represent Cryptococcus diffluens.). The new AFLP genotype, 1B, is indicated in boldface.
1358
NOTES
J. CLIN. MICROBIOL.
TABLE 1. New primer combinations used for mating and serotype determination by PCR
Gene or allele
Primer
MAT␣ serotype A (STE20␣A)
JOHE 7264
JOHE 7265
5⬘-AGCTGATGCTGTGGATTGAATAC-3⬘
5⬘-GTTCAATTAATCTCACTACCTGTAG-3⬘
1,200
JOHE 7267
JOHE 7268
5⬘-ATAGGCTGGTGCTGTGAATTAAG-3⬘
5⬘-GTTCAAGTAATCTCACTACATGCG-3⬘
1,200
JOHE 7270
JOHE 7272
5⬘-ATCAGAGACAGAGGAGGAGCAAGAC-3⬘
5⬘-TCCACTGGCAACCCTGCGAG-3⬘
870
JOHE 7273
JOHE 7275
5⬘-GTTCATCAGATACAGAGGAGTGG-3⬘
5⬘-CTCCACTGTCAAACCTACGGC-3⬘
870
MAT␣ serotype D (STE20␣D)
MATa serotype A (STE20aA)
MATa serotype D (STE20aD)
Size (bp)
from the Northeast region, belonged to AFLP genotypes 1a
and 1d. Isolates from non-AIDS patients isolated from the
Southeast region before the onset of the AIDS pandemic belonged to all five subtypes. Unfortunately, it is not clear
whether strains belonging to AFLP genotypes 1a and 1d preferentially occur in AIDS patients, because all of our isolates
originating from AIDS patients came from a geographically
restricted area (Northeast region). Brazilian populations of C.
neoformans var. grubii differ geographically to some extent,
because AFLP type 1 subtypes 1c and 1e were observed only in
the Southeast states.
Three genotypes occurred within C. gattii (Fig. 1). Interestingly, seven isolates from the Northeast and Southeast states
with serotype B MAT␣ belonged to AFLP genotype 6 (Fig. 1).
The occurrence of AFLP genotype 6, which is similar to genotype VGII of W. Meyer, has only recently been reported
from Brazil (17, 23). Isolates of this genotype are known from
non-AIDS patients and environmental samples—mainly in
North and South America, but also in Northern Australia (2, 4,
17, 23). Because this genotype is known to be implicated in a
recent outbreak of C. gattii on Vancouver Island, Canada (T.
FIG. 2. AFLP patterns of AFLP genotypes 1, 1A, and 1B showing the presence of genetic markers co-occurring in all pairs of these genotypes.
Isolates 125.91, RV64610, RV65662, HamdanC3⬘1, Hamdan229, and RV58146 were included as a reference. The numbers given at the branches
represent the cophenetic values.
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MAT␣ strains and represent a hitherto unknown genotype,
which we labeled AFLP genotype 1B (Fig. 1 and 2). Four of
these isolates came from pigeon droppings collected in São
Paulo and Rio de Janeiro, and one came from a non-AIDS
patient in São Paulo (Southeast region). Genotypes 1, 1A, and
1B share a number of AFLP bands: some bands occur only in
pairs of these genotypes, and a few bands appear to be a
specific genotype (Fig. 2). Hence, unique AFLP markers may
discriminate populations of C. neoformans var. grubii. These
populations may be geographically limited (e.g., isolates of
AFLP genotype 1B from the Southeast states of Brazil) or
limited to different ecological niches (e.g., pigeon droppings
and non-AIDS patients). It would be interesting to identify the
molecular nature of C. neoformans var. grubii markers in order
to understand their distribution and transmission and to assess
a possible function of the corresponding genes, if any, in yeast
biology.
Within the main AFLP cluster 1, five subclusters (labeled 1a
to 1e) could be identified showing 85 to 94% similarity within
the subclusters and 62 to 88% similarity among the subclusters
(data not shown). Isolates from AIDS patients, all originating
Sequence
VOL. 42, 2004
Boekhout et al., unpublished observations), some concern
seems justified with respect to the virulence of this genotype,
because two Brazilian isolates originated from non-AIDS patients.
We thank FAPESP (no. 99/06171-2) for financial support.
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NOTES
Irma Nelly G. Rivera