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CLINICAL MICROBIOLOGY - RESEARCH PAPER
Brazilian Journal of Microbiology
https://doi.org/10.1007/s42770-024-01313-1
Introduction
Cryptococcus neoformans and C. gattii form two species
complexes that are etiological agents of cryptococcosis, a
systemic disease acquired through the inhalation of the fun-
gal cells – desiccated blastoconidia or basidiospores – dis-
persed in the environment [1]. After inhalation, the infection
develops into an initial dormant state and may further prog-
ress into the most common symptom, meningoencephalitis,
with other manifestations such as pneumonia and lesions in
dierent tissues due to its systemic nature [2–4].
The infection with C. neoformans has major signicance
for causing cryptococcosis in immunosuppressed indi-
viduals, with estimates of 152,000 cases of cryptococcal
meningitis and 112,000 worldwide annual deaths in 2020,
responsible for 19% of death in patients with HIV/AIDS
Responsible Editor: Luis Augusto Nero.
Fernanda Cristina de Albuquerque Maranhão
fcam@icbs.ufal.br
Douglas Lyra de Holanda Fonseca
douglaslyra@outlook.com
Denise Maria Wanderlei da Silva
denise.wanderlei@gmail.com
1 Institute of Biomedical Sciences, Department of
Microbiology, University of São Paulo, São Paulo, Brazil
2 Institute of Biological and Health Sciences, Sector of
Microbiology, Laboratory of Clinical Microbiology, Federal
University of Alagoas, Maceió, Alagoas, Brazil
3 Institute of Biological and Health Sciences, Sector of
Microbiology, Laboratory of Clinical Microbiology, Federal
University of Alagoas, Av. Lourival de Melo Mota, S/N,
Tabuleiro do Martins, Maceió 57072-900, Alagoas, Brazil
Abstract
Cryptococcosis is one of the major life-threatening opportunistic/systemic fungal diseases of worldwide occurrence, which
can be asymptomatic or establish pneumonia and meningoencephalitis mainly in immunosuppressed patients, caused by
the Cryptococcus neoformans and C. gattii species complexes. Acquisition is by inhaling fungal propagules from avian
droppings, tree hollows and decaying wood, and the association of the molecular types with geographic origin, virulence
and antifungal resistance have epidemiological importance. Since data on cryptococcosis in Alagoas are limited, we sought
to determine the molecular types of etiological agents collected from clinical and environmental sources. We evaluated 21
isolates previously collected from cerebrospinal uid and from environment sources (pigeon droppings and tree hollows)
in Maceió-Alagoas (Brazil). Restriction fragment length polymorphism of URA5 gene was performed to characterize
among the eight standard molecular types (VNI-VNIV and VGI-VGIV). Among isolates, 66.67% (14) were assigned to
C. neoformans VNI – 12 of them (12/14) recovered from liquor and 2 from a tree hollow (2/14). One isolate from pigeon
droppings (4.76%) corresponded to C. neoformans VNIV, while ve strains from tree hollows and one from pigeon drop-
pings (6, 28.57%) to C. gattii VGII. VNI-type was present in clinical and environmental samples and most C. neoformans
infections were observed in HIV-positive patients, while types VNIV and VGII were prevalent in environmental sources
in Alagoas. This is the rst molecular characterization of Cryptococcus spp. in Alagoas, our study provides additional
information on the ecoepidemiology of Cryptococcus spp. in Brazil, contributing to a closer view of the endemic species.
Keywords Cryptococcus neoformans · Cryptococcus gattii · Cryptococcosis · Molecular typing · RFLP
Received: 3 July 2023 / Accepted: 21 March 2024
© The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia 2024
Molecular characterization of clinical and environmental isolates from
the Cryptococcus neoformans/C. Gattii species complexes of Maceió,
Alagoas, Brazil
Douglas Lyrade Holanda Fonseca1· Denise Maria Wanderlei daSilva2·
Fernanda Cristinade Albuquerque Maranhão3
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Brazilian Journal of Microbiology
[5], while infections caused by C. gattii aect immunocom-
petent patients [6], primarily children and the elderly, show-
ing a primary infection trait and pathogenic aptitude [1, 7].
Recently, the World Health Organization (WHO) (2022)
included C. neoformans in the critical group and C. gattii in
the medium group of the Fungal Priority Pathogens List [8],
to direct research and raise awareness of this fungal disease,
strengthening new actions to control these pathogens.
The two species complexes are subdivided into eight
major molecular types, proposed in 2009 by the Interna-
tional Society for Human and Animal Mycology (ISHAM)
working group [9], recognizable by dierent molecular
techniques, such as PCR-ngerprinting, restriction fragment
length polymorphism (RFLP) of the URA5 gene, ampli-
ed fragment length polymorphism (AFLP), multi-locus
sequence typing (MLST) and whole genome sequencing
(WGS), subdividing C. neoformans in VNI, VNII, VNIII
and VNIV and C. gattii in VGI, VGII, VGIII and VGIV [9–
11]. One of the most applied techniques is the PCR-RFLP
of URA5, a low-cost methodology which benets from the
nucleotide sequence of URA5 in C. neoformans and in C.
gattii diering in only about 8%, showing identical size and
introns position, and having the product (orotidine mono-
phosphate pyrophosphorylase) structure with homology in
98% of the amino acids, demonstrating a recent phyloge-
netic relation between these species [12, 13].
Recently, 3 other molecular types have been identied
(VNB, VGV and VGVI) [14–18], however these demon-
strate low distribution and a smaller frequency compared
to the 8 stablished genotypes [11]. Additionally, a nomen-
clature change has been proposed by Hagen et al. (2015)
[17], moving the molecular types into species level: C. neo-
formans (VNI, VNII), C. neoformans X C. deneoformans
hybrid (VNIII), C. deneoformans (VNIV), C. gattii (VGI),
C. deuterogattii (VGII), C. bacillisporus (VGIII), C. tetra-
gattii (VGIV) and C. decagattii (VGIV/VGIIIc). In face of
this proposal, Kwon-Chung et al. (2017) [19] suggested the
use of “species complexes”, reasoned by the new molecu-
lar types being discovered and the lack of biological dier-
ences among the clades, and later Hagen et al. (2017) [20]
defended their perspective, however there is still no consen-
sus among researchers about this matter, thus we decided
to use “species complexes” in this study following recent
literature [11, 21].
C. neoformans sensu lato occurs in all continents and is
the most common pathogen of cryptococcosis in all regions
of Brazil [22, 23]. It is usually found on avian droppings
(mainly pigeon) and decaying organic material [1, 24]. On
the other hand, C. gattii sensu lato occurs mainly in tropical
and subtropical regions of the world [1], it is endemic in
the northern and northeastern regions of Brazil [22], usu-
ally colonizing decaying wood material and tree hollows
from dierent species [4]. Limited data on cryptococcosis
is still a reality in Latin America [11, 24] and the ecoepi-
demiological data in Brazil are restricted to a few regions,
lacking information on the correlation of the environmental
and clinical isolates, with some states without any molecu-
lar characterization to date [22, 24–26]. Our purpose was to
determine for the rst time the molecular types of clinical
and environmental isolates collected in Maceió (Alagoas,
Brazil) by URA5-RFLP, aiming to update the ecoepidemio-
logical distribution of C. neoformans/C. gattii species com-
plexes in Brazil.
Materials and methods
Fungal isolates
This study evaluated 21 isolates collected from 2013 to
2016, previously identied by the Clinical Microbiology
Laboratory (LMC), Federal University of Alagoas (UFAL),
as Cryptococcus sp. through phenotypic methods (india
ink staining followed by urease and phenoloxidase tests),
these isolates were stocked at the mycological collection of
the LMC (SisGen access no. A180447/A329989). Fourteen
clinical isolates were collected from the cerebrospinal uid
(CSF) of patients with suspected cryptococcal meningoen-
cephalitis assisted at Hospital Escola Doutor Hélvio Auto
(HEHA), besides seven environmental isolates collected
from decaying material in a tree hollow and pigeon drop-
pings located in public squares from Maceió (Alagoas,
Brazil).
Standard strains of each molecular type provided by Insti-
tuto Nacional de Infectologia Evandro Chagas – Fundação
Oswaldo Cruz (INI-Fiocruz) were utilized: C. neoformans
WM 148 (VNI), WM 626 (VNII), WM 628 (VNIII), WM
629 (VNIV) and C. gattii WM 179 (VGI), WM 178 (VGII),
WM 175 (VGIII), WM 779 (VGIV).
Isolate preparation
Collection of the isolates were maintained stocked at -20ºC
after saturated cultures were grown in Sabouraud dextrose
broth, with new stock cultures made every 6 months. For
the use, isolates were inoculated in Sabouraud dextrose agar
(SDA) for the period of 48 h at 30ºC, then approximately 10
µL aliquot of cells were harvested by an inoculation loop
and inserted in 1.5 mL sterilized microtubes, this material
was incubated overnight at -20ºC for mechanical breaking
of the yeast capsule. Alternatively, the isolates were inocu-
lated in 1 mL of YEPD broth (yeast 1%, peptone 2% and
dextrose 1%) with 0.5 M of NaCl, to avoid capsule forma-
tion, in 1.5 mL sterilized microtubes and incubated for 48 h
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Brazilian Journal of Microbiology
at 30ºC in a shaker at 150 rpm until cultures were saturated.
Then, the samples were pelleted by centrifuging in 17,000 x
g for 2 min to collect the cells.
DNA extraction
Genomic DNA was extracted and puried using Wizard®
Genomic DNA Purication Kit (Cat. A1120; Promega,
Madison, USA) or following the methodology described
by Ferrer et al. (2001) [27] and Rede de Criptococose
Brasileira (RCB) [28], with some modications: 10 µL ali-
quot of cells was suspended in 500 µL of lysis buer (0.5%
sodium dodecyl sulfate, 1.4% NaCl, 0.73% dihydrate EDTA
and Tris-HCl 0.2 M, pH = 8.0) and 5 µL of 2-mercapetha-
nol, followed by incubation at 65ºC for 1 h. Then, 500 µL
of phenol: chloroform: isoamyl alcohol (25:24:1) was added
into the tubes and mixed thoroughly for 2 min, followed by
centrifugation for 15 min at 32,000 x g. The supernatant was
transferred to new tubes and mixed with an equal volume of
isopropanol for incubation at -20ºC overnight, aiming the
precipitation of nucleic material. Following, the tubes were
centrifuged at 32,000 x g (15 min) for pellet formation and
removal of the supernatant. The pellet was suspended in 200
µL of 70% ethanol for cleaning and centrifuged at 32,000 x
g (15 min), removing the supernatant. The DNA pellet was
air dried, resuspended in 100 µL of sterile ultrapure water
for treatment with RNase A (Cat. A7973; Promega, Madi-
son, USA) (1 h at 37ºC) and the material was stocked at 4ºC.
Molecular typing by URA5-RFLP
URA5 PCR was performed individually as described by
Meyer et al. 2003 [29], in a nal volume of 50 µL with an
aliquot of genomic DNA, 1X PCR buer (5X Colorless
GoTaq® Flexi Buer, Promega; Cat. M890A), 0.2 mmol
of each dNTP (Cat. DNTP100; Sigma-Aldrich, Burlington,
USA), 3 mmol of magnesium chloride (Cat. A351B; Pro-
mega, Madison, USA), 1.5 U of GoTaq® Flexi DNA poly-
merase (Cat. M829A; Promega, Madison, USA), 50ng of
primers URA5 (5’- A T G T C C T C C C A A G C C C T C G A C T C C
G-3’) and SJ01 (5’- T T A A G A C C T C T G A A C A C C G T A C T
C-3’). The thermocycler (Bio-Rad®, Hercules, USA) was
congured to the following cycles: 2 min at 94 °C for initial
denaturation, followed by 35 cycles of 45 s at 94 °C for
denaturation, 1 min at 61 °C for annealing, 2 min at 72 °C
for extension and a nal cycle at 72 °C for 10 min for nal
extension.
The amplied product was stocked at 4 °C for further
procedures. Products were visualized on a 1.4% agarose gel
with 1X TBE and Nancy-520 (Cat. 01494; Sigma-Aldrich,
Burlington, USA) after electrophoresis with a 100 bp DNA
ladder (Cat. 15,628,019; Invitrogen, Waltham, USA).
According to Meyer et al. (2003) [29], the URA5 ampli-
ed products were double digested by restriction enzymes
Sau96I (5 U/µL; Cat. R0165S; New England Biolabs, Mas-
sachusetts, USA) and HhaI (10 U/µL; Cat. R0139S; New
England Biolabs, Massachusetts, USA) in a nal volume of
30 µL with 1X NEBuer (Cat. B7202; New England Bio-
labs, Massachusetts, USA) for incubation at 37ºC for 3 h.
For evaluation, electrophoresis with an agarose gel (3%)
stained with Nancy-520 was performed and the genotypes
were assigned by comparison with their respective refer-
ence strain.
Brazilian ecoepidemiological map
Articles reporting dierent molecular types of Cryptococ-
cus neoformans/C. gattii species complexes in Brazil were
selected from PubMed and Scielo databases, using the pri-
mary descriptors “molecular type” OR “genotype” AND
“cryptococcus” AND “Brazil” to select articles published
from 2008 to 2024 with information about place, description
of sample and molecular type. After data compilation, we
constructed a Brazilian map using the QGIS v.3.34.3 soft-
ware and the Instituto Brasileiro de Geograa e Estatística
(IBGE) database, separating Cryptococcus molecular types
by states and including the types described in Alagoas.
Results
The clinical isolates (n = 12) were collected from cerebro-
spinal uid of 9 patients that underwent CSF recollection to
monitor the eectiveness of the treatment and remission of
the yeast. Out of the 9 patients with cryptococcal meningo-
encephalitis, 4 were HIV-positive (44.44%), 2 HIV-negative
(22.22%) and 3 had no information about seropositivity in
their medical records (33.34%). The mortality rate was 75%
(3/4) among the HIV positive individuals and 50% (1/2) in
the HIV negative. One HIV-negative patient was released
from medical assistance and 4 patients had no clinical evo-
lution information registered. The environmental strains
(n = 9) were isolated from a tree hollow and from pigeon
droppings (Table 1).
DNA extraction was performed by two methodologies,
the method described by Ferrer et al. (2001) [27] was eec-
tive and allowed the recovery of large amounts of fungal
genomic material. However, the usage of the Wizard Kit
allowed faster and more puried acquisition of the DNA,
being used in most of the samples. URA5 PCR-RFLP of the
21 Cryptococcus spp. clinical and environmental isolates
compared with the 8 molecular reference strains revealed
71.43% (15/21) as C. neoformans sensu lato, whereas
28.57% (6/21) were C. gattii sensu lato. The molecular
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Brazilian Journal of Microbiology
types characterized in Maceió (Alagoas, Brazil) were VNI
(66.67% − 14/21), VGII (28.57% − 6/21) and VNIV (4.76%
− 1/21) (Fig. 1). All clinical isolates (12) corresponded to
genotype VNI, in both HIV-positive and HIV-negative
patients, while the most common molecular type in the 9
environmental isolates were VGII (6/9), followed by VNI
(2/9) and VNIV (1/9). It was possible to identify 2 molecular
types (VNI and VGII) collected from the same tree hollow,
whereas in the pigeon droppings the genotypes identied
were VNIV and VGII.
After compiling the public molecular epidemiological
data of Cryptococcus spp. in Brazil, it was possible to con-
struct an ecoepidemiological map to assess the distribution
of the molecular types in dierent regions (Fig. 2; Table S1).
The genotypes VNI and VGII are the most commomly iden-
tied among the studies, distributed in all regions of Brazil
including the present record in the state of Alagoas, while
VNIV is rarely identied.
Discussion
Cryptococci eradication requires a rapid identication and
eective treatment for the host survival, but in several coun-
tries these measures are not available for the population,
making cryptococcal infection one of the most important
life-threatening opportunistic diseases for AIDS patients [5].
The molecular types of Cryptococcus neoformans and C.
gattii species complexes dier in their ecology, geographi-
cal distribution, pathogenicity and antifungal susceptibility,
even with the phylogenetic proximity and morphological
similarities among the groups [20].
In this study, the isolates had origin in clinical and envi-
ronmental samples from Maceió, Alagoas (northeastern
Brazil), indicating the presence of molecular types VNI,
VNIV and VGII. Studies about the ecoepidemiology of
Cryptococcus spp. should take into account both clinical
and environmental samples for a better understanding of its
geographical distribution, as the infection is disseminated
through borne fungal propagules from the environment and
is also considered as a zoonosis, indicating that molecular
types collected from clinical samples were disseminated in
certain regions [26, 30].
The mortality rate observed in our study was 44.44%
(4/9). However, the rate was higher among HIV-positive
patients (75%; 3/4). Several factors are involved in the
disease mortality, such as delay in diagnosis, the lack of
eective treatment, virulence of the strains and level of
immunosuppression of the patient. In the world, the mortal-
ity rate in individuals with cryptococcosis is high, even in
places with rst-line treatment availability, reaching 40% in
developed countries and up to 70% in low-income countries
Table 1 List of clinical and environmental isolates and their complementary information
Clinical isolates Environmental isolates
Isolate Molecular
type
Patient Gender Age HIV Outcome Sample
source
Local Isolate Molecular
type
Sample source Local
1 VNI 1 M 16 - Death CSF HEHA –
Maceió, AL.
13 VGII Tree hollow Praça São
Vicente
– Centro,
Maceió-AL.
2 VNI 14 VGII
3 VNI 15 VNI
4 VNI 2 M 23 +Death 16 VGII
5 VNI 17 VGII
6VNI 3 M 47 - Released 18 VNI
7 VNI 4 F 29 +Death 19 VGII
8VNI 5 F 14 +Death 20 VNIV Pigeon
droppings
Praça Santa
Rita de Cás-
sia– Farol,
Maceió-AL.
9 VNI 6M 44 NA NA
10 VNI 7 F 47 +NA 21 VGII Pigeon
droppings
Praça São
Vicente
– Centro,
Maceió-AL.
11 VNI 8M 23 NA NA
12 VNI 9 M NA NA NA
M: male; F: female; NA: data not available on their medical records; CSF: cerebrospinal uid; HEHA: Hospital Escola Dr. Helvio Auto
1 3
Brazilian Journal of Microbiology
possibly occur due to virulence and pathogeny dierences
among strains of C. neoformans, expressing genes that
guarantee the ability to cause infections in immunocompe-
tent individuals [1]. Recently, there are also reports of VNI
causing infections in HIV-uninfected patients in Brazil [25,
36, 38–40], this evidence demonstrates the ability of the
molecular type VNI in causing infections to HIV-negative
individuals, as well as in 2 patients from Alagoas.
Considering the environmental samples, the predomi-
nance of each type may vary according to the niche with
which the sampling is made, due to better adaptation that
each genotype has to specic habitats [3, 41]. Tree hollows
are considered to be predominantly colonized by C. gattii
[42], but likewise, this niche is supportive for C. neoformans
to grow [3]. According to Trilles et al. (2003) [43], dierent
molecular types of Cryptococci can inhabit a common tree,
as observed in a study carried out in Teresina (Piauí, Brazil)
and also found in Italy by Cogliati et al. (2020) [4], when
they observed the types VNI and VNIV sharing the same
tree, but with dierent populational density in dierent parts
of it. Barbosa et al. (2013) [44] identied VGI and VNI iso-
lates in tree hollows in Rio de Janeiro, while Costa et al.
(2009) [45] reported VGII and VNI associated with decay-
ing material from a tree hollow in Belém (Pará, Brazil). This
[31]. In Brazil, mortality rate may vary from 26 to 70%,
depending on the evaluated place, treatment availability and
patient immunosuppression [32, 33]. Current data show a
decrease of 28% of deaths related to AIDS in the world from
2014 to 2020 [5], in accordance with the Brazilian Ministry
of Health [34], which has suggested a decrease in individu-
als living with HIV in Brazil, although an increase occurred
in the northern and northeastern regions, where the crypto-
coccal surveillance is at most scarcity.
Even though clinical isolates were from HIV-infected and
HIV-uninfected patients, all of them were characterized as
VNI in our study. This molecular type is characterized as the
primary infecting agent of cryptococcal meningoencephali-
tis in individuals with advanced immunosuppression stage
due to AIDS [1]. This is observed by Tsujisaki et al. (2013)
[35], Favalessa et al. (2014) [36], Aguiar et al. (2017) [32]
and da Silva et al. (2020) [33] that also identied VNI as the
predominant molecular type in infections of HIV-positive
individuals in Mato Grosso do Sul, Mato Grosso, Minas
Gerais and São Paulo (Brazil), respectively.
In immunocompetent patients, the infections are usually
associated with VGII. Despite that, there are evidences of
VNI also infecting these individuals as observed in China,
where VNI is frequently associated [37]. These distinctions
Fig. 1 Characterization of clinical and environmental isolates of Cryp-
tococcus sp. by URA5 PCR-RFLP. Restriction fragment length poly-
morphism (RFLP) prole of URA5 gene double-digested with restric-
tion enzymes Sau96I and HhaI. M: 100 bp DNA ladder (molecular
weight); lanes VNI-VGIV: reference strains; lanes 1–12: clinical iso-
lates; lanes 13–21: environmental isolates; bp: base pairs
1 3
Brazilian Journal of Microbiology
There is a possibility that the pigeon droppings that were
positive for VGII in our study were contaminated with the
strains inhabiting a neighboring tree, approximately 3 m
away – from which we also identied the same molecular
type. This may be considered due to the fact that Crypto-
coccus spp. has already demonstrated great achievement to
disperse to and colonize new habitats [48]. As an example,
there is the report of type VGII described as the infecting
agent in a cryptococcosis outbreak in Vancouver, Canada
[49], demonstrating its ability to adapt to new locations and
be dispersed through dierent means, such as wood export,
air and water currents, as well as biological sources (as
birds and insects), allowing colonization in areas far from
its endemic region [4, 22, 25, 50].
The molecular type VNI occurs worldwide and is the
most prevalent in clinical and environmental samples
[51], with less frequency only in samples collected from
pattern was also observed in our study, with ve VGII iso-
lates and two VNI identied inhabiting a single tree hollow.
An interesting observation is that dierent molecular types
of C. gattii have not been detected in association in the same
location, such as a tree, which may suggest a stronger com-
petition among VG types.
C. neoformans tends to be the dominant species in pigeon
droppings, once it utilizes urea and creatinine from the sub-
strate as nitrogen source [41, 46]. The molecular type VNI
is the most common type in pigeon droppings in Brazil [22,
23]. However, in the present study we did not recover any
strain from this type in pigeon droppings, only 1 VNIV and
1 VGII isolates. C. gattii is unusual in this environment and
can grow without adequate adaptation to inhabit this niche
for long periods [41]. Despite that, Teodoro et al. (2013)
[47] have reported about the recovery of C. gattii in 5.2%
of the identied species from bird droppings in São Paulo.
Fig. 2 Map showing the molecular types distribution of Cryptococcus
neoformans and C. gattii species complexes in Brazil. Data described
in the literature between 2008 and 2024, with the inclusion of molecu-
lar types described for the rst time in Alagoas. Molecular types were
indicated by triangle and circle shapes with dierent colors for each
type. The QGIS v.3.34.3 software and the Instituto Brasileiro de Geo-
graa e Estatística (IBGE) database were used to construct the map.
Abbreviations correspond to each Brazilian state. Reference list: [15,
22, 25, 32, 33, 35, 36, 38–40, 44, 45, 50, 56, 57, 59, 69, 71–116]
1 3
Brazilian Journal of Microbiology
epidemiological control. However, the brand-new fungal
priority pathogens list released by WHO (2022) [8] dem-
onstrates an increased concern about this infection, thus a
gradual expansion in epidemiological information related
to infections by Cryptococcus spp. is expected, with better
surveillance around the world.
Ecological and clinical studies can provide informa-
tion on the epidemiology of these pathogens and charac-
terization of niches favorable for the colonization of these
microorganisms, which represent a risk to the population
when exposed to propagules [30]. Prevention of infection
is hampered by the invasive and disseminative capacity of
these organisms [62], but the chances of exposure can be
minimized through the use of personal protective equipment
(PPE) for respiratory protection in places with high concen-
tration of avian excreta or wood cutting. In cases of cleaning
of contaminated areas, the decontamination and spraying
with water or oil of the material should be previously made
to avoid aerobic propagules [63].
Here we report VNI, VNIV and VGII as prevalent molec-
ular types in Maceió (Alagoas, Brazil), with VNI identied
in clinical and environmental samples, which highlights
dissemination points of this genotype in our area. Although
VNIV and VGII have not been identied in clinical sam-
ples, their presence in the environment is already a determi-
nant for the possibility of infection, since the dispersion of
any genotype occurs from environmental sources and dis-
semination from human to human has not yet been reported
[4, 64]. Despite the fact that transmission by organ dona-
tion [65] and direct contamination of wounds [66] is rarely
reported in humans, there are reports of maternal-fetal trans-
mission in cetaceans [67], demonstrating the great dissemi-
native capacity of these microorganisms, in addition to the
fact that Cryptococcus sp. manages to infect a wide range of
wild and domestic animals, from terrestrial to marine envi-
ronments [68], making constant exposure to contaminated
excreta of avian pets an important mean of zoonotic trans-
mission [69, 70]. Such information leads us to take more
cautious measures regarding the possibility of contamina-
tion in these situations.
The importance of determining molecular types of the
C. neoformans and C. gattii species complexes goes behind
ecoepidemiology, since there are clinically important dier-
ences between each evaluated types, and until our research
there was no information available about the molecular
types of Cryptococcus spp. prevalent in the state of Alagoas.
tree hollows [41], and predominant in cryptococcal infec-
tions in Brazil [22–24, 44]. Considering the environmental
samples from our study, the VNI prevalence was lower than
expected, once this type was not identied on its preferen-
tial niche – pigeon droppings.
The molecular type VGII is the most identied group
in C. gattii infections, distributed primarily in the Ameri-
cas [23, 52] and considered endemic from the northern
and northeastern regions of Brazil [24, 53]. It is collected
mainly from decaying organic matter and is associated with
primary infections [3]. In our study, VGII was the predomi-
nant type among the environmental isolates, identied in
a tree hollow and in pigeon droppings, but not on clinical
samples. As this type is endemic to the northern and north-
eastern regions, its prevalence is usually high, as shown by
Trilles et al. (2008) [22]. VGII was the most prevalent type
(63.3%) in clinical and environmental isolates (n = 107) col-
lected from the states of Roraima, Amazonas, Piauí, Per-
nambuco and Bahia [22].
Molecular type VNIV exhibited low prevalence in our
study, as only one isolate was identied from pigeon drop-
pings, similar to what is described in Brazil [24]. It is dis-
tributed primarily in the European countries, probably due
to its better adaptation to temperate weather [4], and iden-
tied from the environment commonly from pigeon drop-
pings or associated with dierent tree species [52]. It shows
scarce clinical association, being cutaneous cryptococcosis
one of the few clinical manifestations related [54]. There
are reports of this type in tree hollows from Rio Grande do
Sul [55], São Paulo [56], and in a patient from Minas Gerais
[57]. Firacative et al. (2021) [23] reported type VNIV in
6.32% of isolates from Latin America and Trilles et al.
(2008) [22] in 4.7% of the isolates from the northern and
northeastern regions of Brazil.
The most prevalent genotypes demonstrated in this study
are the main agents of opportunistic infections in immu-
nosuppressed individuals (VNI) and primary infections in
healthy people (VGII) [1]. It has been shown that VGII is
more resistant to azoles, as uconazole and itraconazole,
regardless of the original geographic area of the isolate
[58, 59]. According to Trilles et al. (2012) [58], VGII was
more resistant than VNI to uconazole, albaconazole, vori-
conazole, itraconazole, ravuconazole and 5-ucytosine,
being amphotericin B the only antifungal that did not show
response variation from dierent molecular types.
In Brazil, cryptococcosis is still a disease that does not
require compulsory notication [60], even though there are
reports of mortality rate reaching 10.96/million inhabitants
in Mato Grosso, in direct cause of death, or 70.41/million
inhabitants in Santa Catarina, in associated cause of death
[61]. Only a few Brazilian states have measures forcing
the inclusion of cryptococcosis among the diseases with
1 3
Brazilian Journal of Microbiology
References
1. May RC, Stone NRH, Wiesner DL et al (2016) Cryptococcus:
from environmental saprophyte to global pathogen. Nat Rev
Microbiol 14:106–117. https://doi.org/10.1038/nrmicro.2015.6
2. Lin X, Heitman J (2006) The Biology of the Cryptococcus neo-
formans Species Complex. Annu Rev Microbiol 60:69–105.
https://doi.org/10.1146/annurev.micro.60.080805.142102
3. Maziarz EK, Perfect JR (2016) Cryptococcosis. Infect Dis Clin
North Am 30:179–206. https://doi.org/10.1016/j.idc.2015.10.006
4. Cogliati M, Patrizia P, Vincenzo C et al (2020) Cryptococcus neo-
formans species complex isolates living in a tree micro-ecosystem.
Fungal Ecol 44. https://doi.org/10.1016/j.funeco.2019.100889
5. Rajasingham R, Govender NP, Jordan A et al (2022) The global
burden of HIV-associated cryptococcal infection in adults in
2020: a modelling analysis. Lancet Infect Dis 22:1748–1755.
https://doi.org/10.1016/S1473-3099(22)00499-6
6. Henao-Martínez AF, Chastain DB, Franco-Paredes C (2018)
Treatment of cryptococcosis in non-HIV immunocompro-
mised patients. Curr Opin Infect Dis 31:278–285. https://doi.
org/10.1097/QCO.0000000000000458
7. Wanderlei Silva DM, de Albuquerque Maranhao FC (2015) Cur-
rent status of the Diagnostic and Genomics of Cryptococcus
neoformans/C. Gattii Species Complex. Fungal Genomics Biol
05:2–5. https://doi.org/10.4172/2165-8056.1000e118
8. World Health Organization (2022) WHO fungal priority patho-
gens list to guide research, development and public health action.
WHO
9. Meyer W, Aanensen DM, Boekhout T et al (2009) Consensus
multi-locus sequence typing scheme for Cryptococcus neofor-
mans and Cryptococcus gattii. Med Mycol 47:561–570. https://
doi.org/10.1080/13693780902953886
10. Cogliati M, Desnos-Ollivier M, McCormick-Smith I et al (2019)
Genotypes and population genetics of neoformans and gattii spe-
cies complexes in Europe and the mediterranean area. Fungal
Genet Biol 129:16–29. https://doi.org/10.1016/j.fgb.2019.04.001
11. Firacative C, Trilles L, Meyer W (2022) Recent advances in
and cryptococcosis. Microorganisms 10:2022–2024. https://doi.
org/10.3390/microorganisms10010013
12. Chen SCA, Meyer W, Sorrell TC (2014) Cryptococcus gat-
tii infections. Clin Microbiol Rev 27:980–1024. https://doi.
org/10.1128/CMR.00126-13
13. Meyer W, Gilgado F, Ngamskulrungroj P et al (2011) Molecu-
lar typing of the Cryptococcus neoformans/Cryptococcus gattii
species Complex. Cryptococcus: from human pathogen to model
yeast. ASM, p 434
14. Litvintseva AP, Thakur R, Vilgalys R, Mitchell TG (2006) Mul-
tilocus sequence typing reveals three genetic subpopulations of
Cryptococcus neoformans var. Grubii (serotype A), including a
unique population in Botswana. Genetics 172:2223–2238. https://
doi.org/10.1534/GENETICS.105.046672
15. Ferreira-Paim K, Andrade-Silva L, Fonseca FM et al (2017)
MLST-Based Population Genetic Analysis in a global context
reveals clonality amongst Cryptococcus neoformans var. Gru-
bii VNI isolates from HIV patients in Southeastern Brazil. PLoS
Negl Trop Dis 11:e0005223. https://doi.org/10.1371/journal.
pntd.0005223
16. Farrer RA, Chang M, Davis MJ et al (2019) A New Lineage of
Cryptococcus gattii (VGV) discovered in the Central Zambezian
Miombo Woodlands. MBio 10:1–19. https://doi.org/10.1128/
mBio.02306-19
17. Hagen F, Khayhan K, Theelen B et al (2015) Recognition of
seven species in the Cryptococcus gattii/Cryptococcus neofor-
mans species complex. Fungal Genet Biol 78:16–48. https://doi.
org/10.1016/j.fgb.2015.02.009
Conclusions
C. neoformans was the prevalent species in both HIV-
infected and HIV-uninfected patients with cryptococcal
meningoencephalitis from Alagoas, being VNI-type the
most frequent, while among the environmental samples C.
gattii VGII prevailed. The VNI, VNIV and VGII genotypes
are adapted to survive in organic matter associated with tree
hollows and in pigeon excreta in dierent urban areas of
the capital, evidencing that VNI and VGII genotypes may
coexist in the same ecological niche and following the same
pattern of prevalence observed in Brazil and other north-
eastern states. Evidence of VNI strains in both clinical and
environmental samples indicate a potential risk of human
infection, mainly to immunosuppressed individuals.
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/s42770-
024-01313-1.
Acknowledgements The authors would like to acknowledge Tamires
Y. M. dos Santos, Isabelle R. de O. Queiroz and Madson C. M. Caval-
cante for previous sampling and phenotypical identication of the iso-
lates. We thank the Hospital Escola Dr. Hélvio Auto personnel for col-
lecting clinical samples. We also thank Marcia Lazera (INI-Fiocruz)
for the kind donation of the standard molecular types of Cryptococcus
spp.
Authors contributions (CRedIT) Douglas Lyra (Investigation; data cu-
ration; writing – original draft preparation; writing – review and edit-
ing), Denise Wanderlei (Supervision; conceptualization; funding ac-
quisition; methodology; project administration; data curation; writing
– review and editing) and Fernanda Maranhão (Supervision; concep-
tualization; funding acquisition; methodology; project administration;
data curation; writing – review and editing).
Funding This work was supported by Ministério da Saúde do Bra-
sil; Conselho Nacional de Desenvolvimento Cientíco e Tecnológico
(CNPq); Fundação de Amparo à Pesquisa do Estado de Alagoas (FA-
PEAL) [grant number 60030000739/2013] and funding scholarship by
Universidade Federal de Alagoas (UFAL).
Data availability All data generated or analyzed during this study are
included in this published article and its supplementary les.
Declarations
Ethics approval and consent to participate This study was approved
by the Research Ethics Committee (number 19035713.8.0000.5013)
in accordance with the resolution 466/2012 of the National Council of
Health from Brazil. Informed consent was obtained from all individu-
als participants in this study.
Conict of interest The authors declare that they have no conict of
interests to this work.
1 3
Brazilian Journal of Microbiology
35. de Sousa Tsujisaki RA, Paniago AMM, da Costa Lima Júnior MS
et al (2013) First molecular typing of Cryptococcemia-causing
Cryptococcus in Central-West Brazil. Mycopathologia 176:267–
272. https://doi.org/10.1007/s11046-013-9676-6
36. Favalessa OC, de Paula DAJ, Dutra V et al (2014) Molecular
typing and in vitro antifungal susceptibility of Cryptococcus spp
from patients in Midwest Brazil. J Infect Dev Ctries 8:1037–
1043. https://doi.org/10.3855/jidc.4446
37. Fang W, Fa Z, Liao W (2015) Epidemiology of Cryptococcus and
cryptococcosis in China. Fungal Genet Biol 78:7–15. https://doi.
org/10.1016/j.fgb.2014.10.017
38. Mora DJ, Pedrosa AL, Rodrigues V et al (2010) Genotype and
mating type distribution within clinical Cryptococcus neofor-
mans and Cryptococcus gattii isolates from patients with crypto-
coccal meningitis in Uberaba, Minas Gerais, Brazil. Med Mycol
48:561–569. https://doi.org/10.3109/13693780903358317
39. Nascimento E, Barião PHG, von Kress MR Z, et al (2021)
Cryptococcosis by Cryptococcus neoformans/Cryptococcus
gattii species complexes in non-HIV-Infected patients in South-
eastern Brazil. Rev Soc Bras Med Trop 54:1–7. https://doi.
org/10.1590/0037-8682-0169-2021
40. Oliveira EP, Inácio CP, de Freitas JF et al (2022) Tuberculosis
and neurocryptococcosis by Cryptococcus neoformans molecu-
lar type VNI in a non-HIV patient: a comorbidities case report. J
Med Mycol 32. https://doi.org/10.1016/j.mycmed.2021.101213
41. Watkins R, King J, Johnston S (2017) Nutritional requirements
and their importance for virulence of pathogenic Cryptococ-
cus species. Microorganisms 5:65. https://doi.org/10.3390/
microorganisms5040065
42. Ellis DH, Pfeier TJ (1990) Natural habitat of Cryptococcus neo-
formans var. Gattii. J Clin Microbiol 28:1642–1644. https://doi.
org/10.1128/jcm.28.7.1642-1644.1990
43. Trilles L, Lazéra M, Wanke B et al (2003) Genetic characteriza-
tion of environmental isolates of the Cryptococcus neoformans
species complex from Brazil
44. Barbosa GG, Trilles L, Wanke B, Lazéra MS (2013) Cryptococ-
cus Gattii VGI and Cryptococcus neoformans VNI Associated
with Wood Decay in Ficus Hollow Trees in Rio De Janeiro, Bra-
zil. Br Microbiol Res J 3:106–115
45. Costa S do, P, Lazéra M dos, Santos S et al (2009) WRRA,
First isolation of Cryptococcus gattii molecular type VGII and
Cryptococcus neoformans molecular type VNI from envi-
ronmental sources in the city of Belém, Pará, Brazil. Mem
Inst Oswaldo Cruz 104:662–664. https://doi.org/10.1590/
S0074-02762009000400023
46. Nielsen K, De Obaldia AL, Heitman J (2007) Cryptococcus neo-
formans mates on pigeon guano: implications for the realized
ecological niche and globalization. Eukaryot Cell 6:949–959.
https://doi.org/10.1128/EC.00097-07
47. Teodoro VLI, Gullo FPPP, Sardi J (2013) de COJ de CO, Envi-
ronmental isolation, biochemical identication, and antifungal
drug susceptibility of Cryptococcus species. Rev Soc Bras Med
Trop 46:759–764. https://doi.org/10.1590/0037-8682-0025-2013
48. Cogliati M (2021) Global warming impact on the expan-
sion of fundamental niche of Cryptococcus gattii VGI in
Europe. Environ Microbiol Rep 13:375–383. https://doi.
org/10.1111/1758-2229.12945
49. Kidd SE, Hagen F, Tscharke RL et al (2004) A rare genotype of
Cryptococcus gattii caused the cryptococcosis outbreak on Van-
couver Island (British Columbia, Canada). Proc Natl Acad Sci
101:17258–17263. https://doi.org/10.1073/pnas.0402981101
50. Herkert PF, Hagen F, de Oliveira Salvador GL et al (2016) Molec-
ular characterisation and antifungal susceptibility of clinical
Cryptococcus deuterogattii (AFLP6/VGII) isolates from South-
ern Brazil. Eur J Clin Microbiol Infect Dis 35:1803–1810. https://
doi.org/10.1007/s10096-016-2731-8
18. Taverna CG, Bosco-Borgeat ME, Mazza M et al (2020) Fre-
quency and geographical distribution of genotypes and mating
types of Cryptococcus neoformans and Cryptococcus gattii spe-
cies complexes in Argentina. Rev Argent Microbiol 52:183–188.
https://doi.org/10.1016/J.RAM.2019.07.005
19. Kwon-Chung KJ, Bennett JE, Wickes BL et al (2017) The case
for adopting the species Complex nomenclature for the Etiologic
agents of Cryptococcosis. mSphere 2:e00357–e00316. https://
doi.org/10.1128/mSphere.00357-16
20. Hagen F, Lumbsch HT, Arsic Arsenijevic V et al (2017) Impor-
tance of resolving Fungal nomenclature: the case of multiple
pathogenic species in the Cryptococcus Genus. mSphere 2:1–13.
https://doi.org/10.1128/msphere.00238-17
21. Beardsley J, Dao A, Keighley C et al (2023) What’s New in Cryp-
tococcus gattii: from bench to Bedside and Beyond. J Fungi 9:41.
https://doi.org/10.3390/jof9010041
22. Trilles L, Lazéra M dos, Wanke S B, et al (2008) Regional pattern
of the molecular types of Cryptococcus neoformans and Crypto-
coccus gattii in Brazil. Mem Inst Oswaldo Cruz 103:455–462.
https://doi.org/10.1590/S0074-02762008000500008
23. Firacative C, Meyer W, Castañeda E (2021) Cryptococcus neo-
formans and Cryptococcus gattii species complexes in latin
America: a map of molecular types, genotypic diversity, and anti-
fungal susceptibility as reported by the latin American cryptococ-
cal study group. J Fungi 7. https://doi.org/10.3390/jof7040282
24. Firacative C, Lizarazo J, Illnait-Zaragozí MT et al (2018) The sta-
tus of cryptococcosis in Latin America. Mem Inst Oswaldo Cruz
113:1–23. https://doi.org/10.1590/0074-02760170554
25. Martins LMS, Wanke B, Lazéra M dos S, et al (2011) Genotypes
of Cryptococcus neoformans and Cryptococcus gattii as agents of
endemic cryptococcosis in Teresina, Piauí (northeastern Brazil).
Mem Inst Oswaldo Cruz 106:725–730. https://doi.org/10.1590/
S0074-02762011000600012
26. do Carmo FN, de Camargo Fenley J, Garcia MT et al (2022)
Cryptococcus spp. and Cryptococcosis: focusing on the infec-
tion in Brazil. Brazilian J Microbiol 53:1321–1337. https://doi.
org/10.1007/s42770-022-00744-y
27. Ferrer C, Colom F, Frasés S et al (2001) Detection and identica-
tion of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal
DNA typing in ocular infections. J Clin Microbiol 39:2873–2879.
https://doi.org/10.1128/JCM.39.8.2873-2879.2001
28. Júnior JL, Laerte V, Júnior P et al Revista de Medicina e Saúde
de Brasília EDITORIAL Implantação da Rede de Criptococose
Brasil no Distrito Federal - RCB-DF. Rev Med e Saúde Brasília
4–6
29. Meyer W, Castañeda A, Jackson S et al (2003) Molecular typ-
ing of IberoAmerican Cryptococcus neoformans isolates. Emerg
Infect Dis 9:189–195. https://doi.org/10.3201/eid0902.020246
30. Vélez N, Escandón P (2016) Distribution and association between
environmental and clinical isolates of Cryptococcus neofor-
mans in bogotá-Colombia, 2012–2015. Mem Inst Oswaldo Cruz
111:642–648. https://doi.org/10.1590/0074-02760160201
31. Rajasingham R, Smith RM, Park BJ et al (2017) Global burden
of disease of HIV-associated cryptococcal meningitis: an updated
analysis. Lancet Infect Dis 17:873–881. https://doi.org/10.1016/
S1473-3099(17)30243-8
32. Aguiar PADF, Pedroso R, dos Sebastião S BA, et al (2017) The
epidemiology of cryptococcosis and the characterization of Cryp-
tococcus neoformans isolated in a Brazilian University Hospital.
Rev Inst Med Trop Sao Paulo 59:63–69
33. da Silva LBB, Bock D, Klafke GBB et al (2020) Cryptococ-
cosis in HIV-AIDS patients from Southern Brazil: still a major
problem. J Mycol Med 30:101044. https://doi.org/10.1016/j.
mycmed.2020.101044
34. BRASIL. Ministério da Saúde (2020) Boletim Epidemiológico
HIV / Aids | 2020. Secr Vigilância em Saúde 1:68
1 3
Brazilian Journal of Microbiology
68. Danesi P, Falcaro C, Schmertmann LJ et al (2021) Cryptococcus
in wildlife and free-living mammals. J Fungi 7:1–23. https://doi.
org/10.3390/jof7010029
69. Siqueira NP, Favalessa OC, Maruyama FH et al (2022) Domes-
tic birds as source of Cryptococcus deuterogattii (AFLP6/VGII):
potential risk for Cryptococcosis. Mycopathologia 187:103–111.
https://doi.org/10.1007/s11046-021-00601-w
70. Sephton-Clark P, McConnell SA, Grossman N et al (2023) Simi-
lar evolutionary trajectories in an environmental Cryptococcus
neoformans isolate after human and murine infection. Proc Natl
Acad Sci 120:2017. https://doi.org/10.1073/pnas.2217111120
71. Alves GSB, Freire AKL, Bentes A dos S, et al (2016) Molecular
typing of environmental Cryptococcus neoformans/C. Gattii spe-
cies complex isolates from Manaus. Amazonas Brazil Mycoses
59:509–515. https://doi.org/10.1111/myc.12499
72. Jalene Alves M, Sadalla do Nascimento I, Santana Cruz K et al
(2022) Cryptococcosis in HIV/AIDS patients in northern Brazil:
clinical aspects, molecular types and isolation of agents from
environmental samples associated with patients. Trop Med Int
Heal 27:387–396. https://doi.org/10.1111/tmi.13737
73. dos Santos Bentes A, Wanke B, dos Santos Lazéra M et al (2019)
Cryptococcus Gattii VGII isolated from native forest and river in
Northern Brazil. Brazilian J Microbiol 50:495–500. https://doi.
org/10.1007/s42770-019-00066-6
74. Brito-Santos F, Barbosa GG, Trilles L et al (2015) Environmental
isolation of gattii VGII from indoor dust from typical wooden
houses in the deep Amazonas of the Rio Negro basin. PLoS ONE
10:1–11. https://doi.org/10.1371/journal.pone.0115866
75. Brito-Santos F, Trilles L, Firacative C et al (2020) Indoor dust as
a source of virulent strains of the agents of cryptococcosis in the
rio negro micro-region of the Brazilian Amazon. Microorganisms
8:1–11. https://doi.org/10.3390/microorganisms8050682
76. Freire AKL, dos Santos Bentes A, de Lima Sampaio I et al
(2012) Molecular characterisation of the causative agents of
cryptococcosis in patients of a tertiary healthcare facility in the
state of Amazonas-Brazil. Mycoses 55:e145–e150. https://doi.
org/10.1111/j.1439-0507.2012.02173.x
77. Khell Da Silva B, Freire AK, Dos Santos Bentes A et al (2012)
Characterization of clinical isolates of the Cryptococcus neo-
formans-Cryptococcus gattii species complex from the Amazo-
nas State in Brazil. Rev Iberoam Micol 29:40–43. https://doi.
org/10.1016/j.riam.2011.05.003
78. Pinheiro SB, Sousa ES, Cortez ACA et al (2021) Cryptococ-
cal meningitis in non-HIV patients in the State of Amazonas,
Northern Brazil. Brazilian J Microbiol 52:279–288. https://doi.
org/10.1007/s42770-020-00383-1
79. Fernando Silva Rocha D, Cruz KS, da Silva Santos CS et al
(2018) MLST reveals a clonal population structure for Crypto-
coccus neoformans molecular type VNI isolates from clinical
sources in Amazonas, Northern-Brazil. PLoS ONE 13:1–15.
https://doi.org/10.1371/journal.pone.0197841
80. Matos CS, De Souza Andrade A, Oliveira NS, Barros TF (2012)
Microbiological characteristics of clinical isolates of Cryptococ-
cus spp. in Bahia, Brazil: Molecular types and antifungal suscep-
tibilities. Eur J Clin Microbiol Infect Dis 31:1647–1652. https://
doi.org/10.1007/s10096-011-1488-3
81. Ribeiro MA, Ngamskulrungroj P (2008) Molecular characteriza-
tion of environmental Cryptococcus neoformans isolated in Vito-
ria, ES, Brazil. Rev Inst Med Trop Sao Paulo 50:315–320. https://
doi.org/10.1590/S0036-46652008000600001
82. Souza LKHH, Souza Junior AH, Costa CR et al (2010)
Molecular typing and antifungal susceptibility of clinical
and environmental Cryptococcus neoformans species com-
plex isolates in Goiania. Brazil Mycoses 53:62–67. https://doi.
org/10.1111/j.1439-0507.2008.01662.x
51. Gago S, Serrano C, Alastruey-Izquierdo A et al (2017) Molecu-
lar identication, antifungal resistance and virulence of Cryp-
tococcus neoformans and Cryptococcus deneoformans isolated
in Seville. Spain Mycoses 60:40–50. https://doi.org/10.1111/
myc.12543
52. Cogliati M (2013) Global Molecular Epidemiology of Cryp-
tococcus neoformans and Cryptococcus gattii: an Atlas of the
molecular types. Scientica (Cairo) 2013:1–23. https://doi.
org/10.1155/2013/675213
53. Casadevall A, Freij JB, Hann-Soden C, Taylor J (2017) Conti-
nental Drift and Speciation of the Cryptococcus neoformans and
Cryptococcus gattii Species Complexes. mSphere 2:e00103-17-6
54. Cogliati M, Zani A, Rickerts V et al (2016) Multilocus sequence
typing analysis reveals that Cryptococcus neoformans var. Neo-
formans is a recombinant population. Fungal Genet Biol 87:22–
29. https://doi.org/10.1016/j.fgb.2016.01.003
55. Medeiros Ribeiro Â, Silva LKRE, Silveira Schrank I et al (2006)
Isolation of Cryptococcus neoformans var. Neoformans sero-
type D from Eucalypts in South Brazil. Med Mycol 44:707–713.
https://doi.org/10.1080/13693780600917209
56. Araújo Mjaneck, Marinho M (2023) Diversity of potencial patho-
genic Cryptococcus species isolated from environment in coun-
try side São Paulo state, Brazil. Peer Rev 5:25–41. https://doi.
org/10.53660/744.prw1920b
57. Andrade-Silva LE, Ferreira-Paim K, Ferreira TB et al (2018)
Genotypic analysis of clinical and environmental Cryptococcus
neoformans isolates from Brazil reveals the presence of VNB
isolates and a correlation with biological factors. PLoS ONE
13:e0193237. https://doi.org/10.1371/journal.pone.0193237
58. Trilles L, Meyer W, Wanke B et al (2012) Correlation of anti-
fungal susceptibility and molecular type within the Cryptococcus
neoformans/C. Gattii species complex. Med Mycol 50:328–332.
https://doi.org/10.3109/13693786.2011.602126
59. Grizante Barião PH, Tonani L, Cocio TA et al (2020) Molecu-
lar typing, in vitro susceptibility and virulence of Cryptococcus
neoformans/Cryptococcus gattii species complex clinical isolates
from south-eastern Brazil. Mycoses 63:1341–1351. https://doi.
org/10.1111/myc.13174
60. BRASIL. Ministério da Saúde (2020) Portaria No 264, De 17 De
Fevereiro De 2020. Diário Of. da União 17–19
61. Alves Soares E, Lazera M, dos Wanke S B, et al (2019) Mortal-
ity by cryptococcosis in Brazil from 2000 to 2012 a descriptive
epidemiological study. PLoS Negl Trop Dis 13:1–17. https://doi.
org/10.1371/journal.pntd.0007569
62. Ribeiro N, de Costa Q, Magalhães MC TFF, et al (2017)
Atorvastatin as a promising anticryptococcal agent. Int J
Antimicrob Agents 49:695–702. https://doi.org/10.1016/j.
ijantimicag.2017.04.005
63. Giro A (2021) Review on Cryptococcus Disease. J Trop Dis Pub-
lic Heal 9:1–6
64. Howard-Jones AR, Sparks R, Pham D et al (2022) Pulmo-
nary cryptococcosis. J Fungi 8:1156. https://doi.org/10.3390/
jof8111156
65. Kaul DR, Vece G, Blumberg E et al (2021) Ten years of donor-
derived disease: a report of the disease transmission advisory
committee. Am J Transpl 21:689–702. https://doi.org/10.1111/
ajt.16178
66. Revenga F, Paricio JF, Merino FJ et al (2002) Primary cutaneous
cryptococcosis in an Immunocompetent host: Case Report and
Review of the literature. Dermatology 204:145–149. https://doi.
org/10.1159/000051835
67. Norman SA, Raverty S, Zabek E et al (2011) Maternal–fetal
transmission of Cryptococcus gattii in Harbor Porpoise. Emerg
Infect Dis 17:304–305. https://doi.org/10.3201/eid1702.101232
1 3
Brazilian Journal of Microbiology
98. Dal Pupo HD, Sena BAG, Reis FCG et al (2019) Polysaccha-
ride diversity in VNI isolates of Cryptococcus neoformans from
Roraima, Northern Brazil. Fungal Biol 123:699–708. https://doi.
org/10.1016/j.funbio.2019.06.003
99. Cardoso PHM, Baroni FDA, Silva EG et al (2013) Feline Nasal
Granuloma due to Cryptoccocus gattii type VGII. Mycopatholo-
gia 176:303–307. https://doi.org/10.1007/s11046-013-9686-4
100. Castro e Silva DMM, Santos DC, CS, Martins MAA et al (2016)
First isolation of Cryptococcus neoformans genotype VNI MAT-
alpha from wood inside hollow trunks of Hymenaea courbaril.
Med Mycol 54:97–102. https://doi.org/10.1093/mmy/myv066
101. FIGUEIREDO TP, LUCAS RC de CAZZANIGARA et al (2016)
Antifungal susceptibility testing and genotyping characterization
of cryptococcus neoformans and gattii isolates from hiv-infected
patients of Ribeirão Preto, São Paulo, Brazil. Rev Inst Med Trop
Sao Paulo 58. https://doi.org/10.1590/S1678-9946201658069
102. Nascimento E, Vitali LH, Tonani L et al (2016) Refractory and/
or relapsing Cryptococcosis Associated with Acquired Immune
Deciency Syndrome: clinical features, genotype, and viru-
lence factors of Cryptococcus spp. Isolates. Am J Trop Med Hyg
94:975–981. https://doi.org/10.4269/ajtmh.15-0595
103. da Silva EC, Guerra JM, Torres LN et al (2017) Cryptococcus
Gattii molecular type VGII infection associated with lung dis-
ease in a goat. BMC Vet Res 13:4–9. https://doi.org/10.1186/
s12917-017-0950-6
104. Ponzio V, Chen Y, Rodrigues AM et al (2019) Genotypic diversity
and clinical outcome of cryptococcosis in renal transplant recipi-
ents in Brazil. Emerg Microbes Infect 8:119–129. https://doi.org/
10.1080/22221751.2018.1562849
105. de Abreu DPB, Machado CH, Makita MT et al (2017) Intesti-
nal Lesion in a dog due to Cryptococcus gattii type VGII and
review of published cases of Canine Gastrointestinal Cryptococ-
cosis. Mycopathologia 182:597–602. https://doi.org/10.1007/
s11046-016-0100-x
106. Silva DC, Martins MA, Szeszs MW et al (2012) Susceptibility to
antifungal agents and genotypes of Brazilian clinical and environ-
mental Cryptococcus gattii strains. Diagn Microbiol Infect Dis
72:332–339. https://doi.org/10.1016/j.diagmicrobio.2011.11.016
107. de Sousa HR, de Oliveira GP, Frazão S, de O et al (2022) Faster
Cryptococcus Melanization increases virulence in experimental
and human cryptococcosis. J Fungi 8. https://doi.org/10.3390/
jof8040393
108. Favalessa OC, Lázera M, dos S, Wanke B et al (2014) Fatal
Cryptococcus gattii genotype AFLP6/VGII infection in a HIV-
negative patient: case report and a literature review. Mycoses
57:639–643. https://doi.org/10.1111/myc.12210
109. de Faria Ferreira M, Brito-Santos F, Henrique Nascimento The-
odoro P et al (2022) Mixed infection by Cryptococcus neoformans
and Cryptococcus gattii and coinfection with paracoccidioido-
mycosis in PLHIV. Med Mycol Case Rep 35:48–50. https://doi.
org/10.1016/j.mmcr.2022.01.006
110. Lomes NR, De Carvalho Melhem MS, Szeszs MW et al (2016)
Cryptococcosis in non-HIV/non-transplant patients: a Brazilian
case series. Med Mycol 54:669–676. https://doi.org/10.1093/
mmy/myw021
111. Maciel RA, Ferreira LS, Wirth F et al (2017) Corticosteroids for
the management of severe intracranial hypertension in menin-
goencephalitis caused by Cryptococcus gattii: a case report and
review. J Mycol Med 27:109–112. https://doi.org/10.1016/j.
mycmed.2016.09.003
112. Silva LM, Ferreira WA, Filho RAAB et al (2020) New ST623 of
Cryptococcus neoformans isolated from a patient with non-hodg-
kin’s lymphoma in the Brazilian Amazon. Ann Clin Microbiol
Antimicrob 19:1–5. https://doi.org/10.1186/s12941-020-00361-3
113. Nascimento E, Bonifácio da Silva MEN, Martinez R, Von
Zeska Kress MR (2014) Primary cutaneous cryptococcosis in an
83. Anzai MC, Dos Santos Lazéra M, Wanke B et al (2014) Cryp-
tococcus Gattii VGII in a Plathymenia reticulata hollow in
Cuiabá, Mato Grosso, Brazil. Mycoses 57:414–418. https://doi.
org/10.1111/myc.12177
84. Maruyama FH, de Paula DAJ, de Menezes I G, et al (2019)
Genetic diversity of the Cryptococcus Gattii Species Complex in
Mato Grosso State, Brazil. Mycopathologia 184:45–51. https://
doi.org/10.1007/s11046-018-0313-2
85. Nunes J, de O, Tsujisaki RA, de Nunes S, de O M et al (2018)
Cryptococcal meningitis epidemiology: 17 years of experience in
a state of the Brazilian pantanal. Rev Soc Bras Med Trop 51:485–
492. https://doi.org/10.1590/0037-8682-0050-2018
86. Andrade-Silva L, Ferreira-Paim K, Silva-Vergara ML, Pedrosa
AL (2010) Molecular characterization and evaluation of viru-
lence factors of Cryptococcus laurentii and Cryptococcus neo-
formans strains isolated from external hospital areas. Fungal Biol
114:438–445. https://doi.org/10.1016/j.funbio.2010.03.005
87. Damasceno-Escoura AH, de Souza ML, de Oliveira Nunes F et al
(2019) Epidemiological, clinical and Outcome aspects of patients
with Cryptococcosis caused by Cryptococcus gattii from a non-
endemic area of Brazil. Mycopathologia 184:65–71. https://doi.
org/10.1007/s11046-018-0304-3
88. Ferreira-Paim K, Andrade-Silva L, Mora DJ et al (2011) Geno-
typing of Cryptococcus neoformans isolated from captive birds
in Uberaba, Minas Gerais, Brazil. https://doi.org/10.1111/j.1439-
0507.2010.01901.x. Mycoses 54:
89. Leão CA, Ferreira-Paim K, Andrade-Silva L et al (2011) Primary
cutaneous cryptococcosis caused by Cryptococcus gattii in an
immunocompetent host. Med Mycol 49:352–355. https://doi.org/
10.3109/13693786.2010.530697
90. Headley SA, Di Santis GW, de Alcântara BK et al (2015) Cryp-
tococcus Gattii-Induced infections in Dogs from Southern Bra-
zil. Mycopathologia 180:265–275. https://doi.org/10.1007/
s11046-015-9901-6
91. Lugarini C, Goebel CS, Condas LAZ et al (2008) Cryptococcus
neoformans isolated from passerine and psittacine bird excreta
in the state of Paraná. Brazil Mycopathologia 166:61–69. https://
doi.org/10.1007/s11046-008-9122-3
92. dos Santos WRA, Meyer W, Wanke B et al (2008) Primary
endemic cryptococcosis gattii by molecular type VGII in the state
of Pará, Brazil. Mem Inst Oswaldo Cruz 103:813–818. https://
doi.org/10.1590/S0074-02762008000800012
93. Brito-Santos F, Reis RS, Coelho RA et al (2019) Cryptococ-
cosis due to Cryptococcus Gattii VGII in Southeast Brazil: the
One Health approach revealing a possible role for domestic
cats. Med Mycol Case Rep 24:61–64. https://doi.org/10.1016/j.
mmcr.2019.04.004
94. Pinto Junior VL, Pone MV da, Pone S et al (2010) SM, Cryp-
tococcus gattii molecular type VGII as agent of meningitis in
a healthy child in Rio de Janeiro, Brazil: report of an autoch-
thonous case. Rev Soc Bras Med Trop 43:746–748. https://doi.
org/10.1590/S0037-86822010000600032
95. Reis RS, Bonna ICF, Antonio IM da S, et al (2021) Cryptococcus
neoformans VNII as the Main cause of cryptococcosis in domes-
tic cats from Rio De Janeiro, Brazil. J Fungi 7:980. https://doi.
org/10.3390/jof7110980
96. Vechi HT, Theodoro RC, de Oliveira AL et al (2019) Invasive fun-
gal infection by Cryptococcus neoformans var. Grubii with bone
marrow and meningeal involvement in a HIV-infected patient:
a case report. BMC Infect Dis 19:1–8. https://doi.org/10.1186/
s12879-019-3831-8
97. Wirth F, Azevedo MI, Goldani LZ (2018) Molecular types of
Cryptococcus species isolated from patients with cryptococcal
meningitis in a Brazilian tertiary care hospital. Brazilian J Infect
Dis 22:495–498. https://doi.org/10.1016/j.bjid.2018.11.002
1 3
Brazilian Journal of Microbiology
116. Vilas-Boas AM, Andrade-Silva LE, Ferreira-Paim K et al (2020)
High genetic variability of clinical and environmental Cryptococ-
cus gattii isolates from Brazil. Med Mycol 58:1126–1137. https://
doi.org/10.1093/mmy/myaa019
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immunocompetent patient due to Cryptococcus Gattii molecular
type VGI in Brazil: a case report and review of literature. Myco-
ses 57:442–447. https://doi.org/10.1111/myc.12176
114. de Carvalho Santana R, Schiave LA, dos Santos Quaglio AS et
al (2017) Fluconazole non-susceptible Cryptococcus neofor-
mans, Relapsing/Refractory cryptococcosis and long-term use of
liposomal amphotericin B in an AIDS patient. Mycopathologia
182:855–861. https://doi.org/10.1007/s11046-017-0165-1
115. Souto ACP, Bonetti LX, Ferreira-Paim K et al (2016) Popula-
tion Genetic Analysis reveals a high genetic diversity in the Bra-
zilian Cryptococcus gattii VGII Population and shifts the global
origin from the Amazon Rainforest to the semi-arid Desert in the
Northeast of Brazil. PLoS Negl Trop Dis 10:e0004885. https://
doi.org/10.1371/journal.pntd.0004885
1 3
