Resistance of Asian Cryptococcus neoformans Serotype A Is Confined to Few Microsatellite Genotypes
et al. (2012) Resistance of Asian Cryptococcus neoformans Serotype A Is Confined to Few
Microsatellite Genotypes. PLoS ONE 7(3): e32868. doi:10.1371/journal.pone.0032868
Resistance of Asian Cryptococcus neoformans Serotype A Is Confined to Few Microsatellite Genotypes
Saad J. Taj-Aldeen
Johan W. Mouton
Jacques F. Meis
Corne H. W. Klaassen
Anastasia P. Litvintseva, Duke University Medical Center, United States of America
Background: Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries. Methodology/Principal Findings: Genetic diversity of Asian isolates of C. neoformans was determined using microsatellite analysis with nine microsatellite markers. The analysis revealed eight microsatellite complexes (MCs) which showed different distributions among geographically defined populations. A correlation between MCs and HIV-status was observed. Microsatellite complex 2 was mainly associated with isolates from HIV-negative patients, whereas MC8 was associated with those from HIV-positive patients. Most isolates were susceptible to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, but 17 (3.4%) and 10 (2%) were found to be resistant to 5-flucytosine and fluconazole, respectively. Importantly, five Indonesian isolates (approximately 12.5% from all Indonesian isolates investigated and 1% from the total studied isolates) were resistant to both antifungals. The majority of 5-flucytosine resistant isolates belonged to MC17. Conclusions: The findings showed a different distribution of genotypes of C. neoformans var. grubii isolates from various countries in Asia, as well as a correlation of the microsatellite genotypes with the original source of the strains and resistance to 5-flucytosine.
Funding: This work was supported by grants (no. 30970130 and no. 80171335) from the National Natural Science Foundation of China. FH was funded by the
Odo van Vloten Foundation, The Netherlands. KK was granted by University of Phayao, Thailand. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: JFM has been a consultant to Astellas, Basilea, Merck and Schering-Plough and received speakers fees from Gilead, Janssen
Pharmaceutica, Merck, Pfizer, and Schering-Plough. CHK received a grant from Pfizer. RW is currently receiving a grant from IIR-Pfizer for doing research on
Cryptococcus. RW is a speaker for Pfizer and Astellas Pharma. All other authors: no potential conflicts of interest relating to employment, consultancy, patents,
products in development or marketed products. The sponsors of the research played no decision-making role in the design, execution, analysis and reporting of
the research. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials.
. These authors contributed equally to this work.
Cryptococcus neoformans and C. gattii are encapsulated
basidiomycetous yeasts that can cause life-threatening infections in humans.
According to the current classification, C. neoformans consists of two
varieties, namely variety grubii (serotype A) and variety neoformans
(serotype D). C. neoformans and C. gattii differ in ecology,
biochemistry, molecular characteristics, and their ability to cause
disease [1,2]. C. neoformans is known as an opportunistic pathogen
because it mainly infects immunocompromised patients, whereas
C. gattii is considered a primary pathogen that infects otherwise
healthy individuals . Clinical characteristics of C. neoformans and
C. gattii differ as well. The latter species causes more frequently
cryptococcomas and has a lower susceptibility to antifungal agents,
resulting in prolonged treatment and a higher mortality rate when
compared to C. neoformans .
Cryptococcus neoformans continues to be the most important cause
of fungal meningitis in immunocompromised patients. The global
burden of cryptococcal infections among HIV-infected patients is
estimated at nearly one million new cases per year . In South
Asia and Southeast Asia, the incidence of HIV infection is the
second-highest with over 4.5 million HIV-infected patients. The
prevalence of cryptococcal meningitis was estimated to be 13.6
and 120 per thousand HIV-infected individuals per year among
HIV-infected patients in these two regions, respectively . In
contrast, most cases of cryptococcosis in East Asia, especially in
China and Japan, where HIV prevalence is low, have been
reported from apparently immuncompetent patients and were
mostly caused by C. neoformans var. grubii . Similarly,
Vietnamese patients with cryptococcal meningitis were usually
infected by this variety, but here it manifests in both
immunocompromised and immunocompetent patients .
Most cryptococcal meningitis cases in Asia are caused by C.
neoformans . Notwithstanding the clinical importance of the
fungus in this part of the world, a systematic survey on the genetic
diversity of the pathogen has not been performed. Genotyping of
isolates may reveal differences in host range and clinical
symptoms. Recently, a genotyping study of Vietnamese clinical
isolates using amplified fragment length polymorphism (AFLP)
revealed two genotypes, VNIc and VNId with the former as the
major genotype for isolates originating from non-HIV-infected
patients . For epidemiological studies of the C. neoformans/C.
gattii complex, several molecular methods have been used, such as
AFLP, M13-based PCR fingerprinting, Multi-locus Sequence
Typing (MLST) and analysis of the intergenic spacer (IGS)
ribosomal DNA sequences .
Microsatellite analysis is a genotyping technique that is
becoming increasingly popular for molecular typing of medically
important fungi . Microsatellites, also referred to as short
tandem repeats (STRs), are genomic sequences that consist of
tandem repeated short motifs . Mutations in microsatellites
lead to a change in the number of repeats creating genotypic
diversity. In some fungi, e.g. Aspergillus fumigatus and A. flavus,
molecular typing using microsatellites proved to be more
discriminatory than MLST [18,19]. In our study, microsatellite
typing was applied to estimate the extent of genetic diversity and
the epidemiological relationships of a collection of clinical
cryptococcal isolates that originated from East, South and
Southeast Asia, and the Middle East. Moreover, a set of
environmental isolates from Japan and Thailand was included.
In vitro antifungal susceptibility was determined for amphotericin
B, flucytosine, fluconazole, itraconazole, voriconazole,
posaconazole and isavuconazole. The aims of this study were: (i) to analyze
the genotypic diversity as well as the distribution of C. neoformans
var. grubii from different geographical regions in Asia, (ii) to relate
the genetic background of the cryptococcal isolates to disease
status and origin from the human body, (iii) to test the in vitro
antifungal susceptiblity of the isolates against seven antifungal
drugs, and (iv) to determine if differences in susceptibility correlate
with the observed genotypic diversity.
Materials and Methods
Isolates and media
A total of 426 clinical isolates of Cryptococcus neoformans var. grubii
isolates were obtained from the collections of the Chinese
Cryptococcus Reference Centre at the Second Military Medical
University, Shanghai, China (n = 115); Department of
Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai,
Thailand (n = 79); Department of Medical Microbiology,
Postgraduate Institute of Medical Education and Research,
Chandigarh, India (n = 61); Sappasitthiprasong Hospital, Ubon
Ratchathani, Thailand (n = 59); Department of Parasitology, Faculty of
Medicine, University of Indonesia, Jakarta, Indonesia (n = 40);
Department of Microbiology, Meiji Pharmaceutical University,
Tokyo, Japan (n = 28); Department of Microbiology, Faculty of
Medicine, Khon Kaen University, Khon Kaen, Thailand (n = 20);
Department of Microbiology, Faculty of Medicine, Health
Sciences Centre, Kuwait University, Jabriya, Kuwait (n = 10);
Mycology Unit, Microbiology Division, Department of
Laboratory Medicine and Pathology, Hamad Medical Corporation, Doha,
Qatar (n = 5) and Department of Pathology, Faculty of Medicine,
Prince of Songkla University, Hat Yai , Thailand (n = 9) (Table
S1). Furthermore, 67 environmental isolates were obtained from
the Department of Microbiology, Faculty of Medicine, Chiang
Mai University, Thailand (n = 58) and the Department of
Microbiology, Meiji Pharmaceutical University, Tokyo, Japan
(n = 9) (Table S2). Two-hundred and thirty-six isolates originated
from HIV-infected patients and 156 isolates were obtained from
HIV-negative patients. Thirty-four out of 426 clinical isolates were
from patients with unknown HIV status.
Species identification was initially performed by standard
mycological methods . L-Canavanine-glycine-bromothymol
blue (CGB) medium was used to distinguish between C. neoformans
and C. gattii isolates . Cryptococcus isolates were stored in sterile
2 ml screw-capped tubes containing porous beads (Microbank,
ProLab Diagnostics, Richmond Hill, ON, Canada) at 280uC until
Mating- and serotype analysis by PCR, and microsatellite
Genomic DNA extraction was performed as previously
described . All PCR amplifications for mating- and serotyping
were carried out in a total volume of 20 ml containing 0.1 mM
dNTPs, 0.5 U of Taq DNA polymerase (Gentaur, Brussels,
Belgium), 1 ml of template genomic DNA (100 ng/ml) and
0.5 mM of the forward and reverse primers, as described by
Bovers et al.  in 16 PCR reaction buffer containing 50 mM
Microsatellite analysis was performed using nine microsatellite
markers . However, instead of three multiplex PCRs, each
locus was amplified in a separate PCR reaction and reaction
products containing different fluorescent labels were pooled prior
to analysis. One microliter of combined reaction product was
mixed with 8.75 ml water and 0.25 ml ET-550R ROX Size
Standard (GE Healthcare, Diegem, Belgium). Samples were
analyzed on a MegaBACE 500 automated DNA analysis platform
(GE Healthcare) equipped with a 48 capillary array according to
the instructions of the manufacturer. Repeat numbers were
assigned using Fragment Profiler v1.2 (GE Healthcare), imported
into BioNumerics v6.0 software (Applied Maths, Sint
MartensLatem, Belgium) and analyzed using the multistate categorical
similarity coefficient. According to previously described criteria,
microsatellite complexes (MCs) were defined as groups of two or
more microsatellite genotypes that differ by a maximum of two
Antifungal susceptibility testing
In vitro antifungal susceptibility testing of amphotericin B (AMB;
Bristol Myers Squibb, Woerden, The Netherlands), flucytosine
(5FC; Valeant Pharmaceuticals, Zoetermeer, The Netherlands),
fluconazole (FLU; Pfizer Central Research, Sandwich, Kent,
United Kingdom), itraconazole (ITR; Janssen Cilag, Tilburg, The
Netherlands), posaconazole (POS; Schering-Plough Corp.,
Kenilworth, NJ, USA), voriconazole (VOR; Pfizer Central Research)
and isavuconazole (ISA; Basilea Pharmaceutica, Basel,
Switzerland) was performed using the standard broth microdilution
method as described in CLSI document M27-A3 . The
minimal inhibitory concentrations (MIC) were read optically after
72 h of incubation at 35uC. For AMB, the MIC was defined as the
lowest concentration of drug showing no yeast growth. For the
other antifungal compounds, the MIC was defined as the lowest
concentration that caused a prominent reduction of yeast growth
($50%). Candida krusei ATCC6258 and Candida parapsilosis
ATCC22019 were used as quality controls.
The resistance breakpoints of FLU and 5FC were taken from
CLSI document M27-A3  and Perfect et al.  as follows:
$16 mg/ml for FLU; $32 mg/ml for 5FC. According to Nguyen
and Yu  the resistance breakpoint of AMB is $2 mg/ml.
Interpretive criteria of ITR and the new azoles have not been
proposed yet for C. neoformans. However, a MIC#1 mg/ml was
suggested as the susceptibility breakpoint for ITR, VOR, POS and
The Simpsons index of diversity was calculated to assess the
genotypic diversity of microsatellite genotypes among the different
populations . The value obtained is scaled from zero to one,
where a value of one indicates that all isolates are different, and a
value of zero means that all belong to the same genotype.
Chi-square and Fisher exact tests were applied to examine the
correlation between MCs and isolate categories, including HIV
status, sample sources (environmental or clinical), and
geographical origin. In vitro antifungal susceptibility testing results were
statistically analyzed using the t-test method. P values less than
0.05 were considered significant for all statistical methods. The
statistics were analyzed using StatsDirect v2.7.8 (StatsDirect,
Cheshire, United Kingdom).
Mating-type and serotype analysis
Of the 426 clinical C. neoformans var. grubii isolates obtained from
HIV-positive patients (n = 236), HIV-negative patients (n = 156),
and those with an unknown HIV status (n = 34), 425 isolates
(99.8%) belonged to mating-type a and serotype A (aA), and one
isolate (0.2%) from a HIV-negative patient in China belonged to
mating-type a and serotype AD (aAaD). All 67 environmental
isolates of C. neoformans var. grubii obtained from avian droppings
from Chiang Mai, Thailand (n = 58) and Tokyo, Japan (n = 9) were
mating type a and serotype A (Table S1 and Table S2).
The genetic diversity of 492 isolates of C. neoformans var. grubii
(aA) and one aAaD hybrid isolate was analyzed by microsatellite
typing. Within this collection of 493 isolates, 265 different
genotypes were found. These 265 genotypes were distributed over
eight microsatellite complexes (MCs), which were defined as
described previously . Of the eight observed MCs, five were
previously found in a collection of cryptococcal isolates from Cuba
(i.e., MC1, MC2, MC3, MC8 and MC12), while three novel MCs
were observed in the Asian collection presented here. These were
assigned MC15, MC16, and MC17 (Figure 1A, Table S1 and
Table S2). Twenty-four of the 493 isolates, which belonged to 19
microsatellite genotypes, could not be assigned to a MC. MC8 was
the predominant MC among Asian isolates (n = 206, 41.8%),
followed by MC2 (n = 151, 30.6%), MC3 (n = 42, 8.5%), MC16
(n = 21, 4.3%), MC17 (n = 15, 3.1%), MC1 (n = 14, 2.8%), MC12
(n = 13, 2.6%) and MC15 (n = 11, 2.2%). The distribution of MCs
was found to differ in each country (Figures 1A and B, and
Table 1). MC2 was predominant among the Chinese, Japanese,
and Thai cryptococcal populations, whereas MC8 was the major
type among isolates of patients from Southeast Asian countries
(i.e., Indonesia and Thailand). In India, the majority of isolates
belonged to MC1 and MC3. In addition, MC16 and MC17 were
the predominant MCs containing Japanese and Indonesian
isolates, respectively. The 17 isolates studied from the Middle
East (Kuwait and Qatar) occur more scattered and belonged to
MC2, MC3, MC8, and MC15 (Figures 1A and B, and Table 1).
The relationship between most MCs is not well resolved.
However, MC2 and MC16 that contained isolates from the
Chinese, Thai, and Japanese populations are well interconnected.
The genotypic diversity of C. neoformans var. grubii was found to
be most diverse (D = 1.000) in the Kuwait and Qatar populations,
followed by the Japanese (D = 0.998), Indonesian (D = 0.994),
Indian (D = 0.983), Chinese (D = 0.975), and Thai populations
(D = 0.968) (Table 2). The genetic diversity within MCs showed
values of Simpsons index of diversity close to 1, thus indicating
that each MC contained a high level of genetic diversity. A
significant correlation between MCs and HIV-status was observed
by the Chi-square (p,0.0001) and Fisher exact tests (p,0.0001)
(Figures 1A and C, and Table 3). The majority of isolates from
HIV-negative patients belonged to MC2 that accounted for 104
out of 156 isolates from HIV-negative patients (66.6%), followed
by MC16 (n = 13, 8.3%). In contrast, MC8 was the predominant
MC containing isolates from HIV-positive patients (138 of 236,
58.5%), followed by MC2 (n = 30, 12.7%), MC3 (n = 33, 14%) and
MC17 (n = 13, 5.5%).
The distribution of environmental and clinical isolates among
MCs was studied using two populations from Tokyo, Japan and
Chiang Mai, Thailand. In both cases, the clinical and
environmental isolates belonged to the same MCs (Figure 1D and Table 4).
MC2 and MC8 were the common MCs in the Thai population.
MC8 contained 26 out of 40 clinical isolates and 45 out of 58
environmental isolates, whereas MC2 contained 12 out of 40
clinical isolates and 8 out of 58 environmental isolates. Among the
Japanese isolates, MC16 contained 16 out of 28 clinical isolates
and 4 out of 9 environmental isolates, and MC2 contained 11 out
of 28 clinical isolates and 3 out of 9 environmental isolates. The
combined results of the Thai and Japanese populations showed a
correlation between MCs and the clinical or environmental origin
(p = 0.0004, Chi-square; p = 0.0001, Fisher exact test). However,
no such correlation was observed within the Thai population from
The predominant MCs in each country are indicated in bold.
Chiang Mai (p = 0.0963, Chi-square; p = 0.0729, Fisher exact test)
nor within the Japanese population from Tokyo (p = 0.9192,
Chisquare; p.0.9999, Fisher exact test).
In vitro antifungal susceptibility testing
The MIC values of all C. neoformans var. grubii isolates for the
seven antifungal compounds tested are listed in Table 5. Almost all
cryptococcal isolates were susceptible to AMB, ITR, FLU, VOR,
POS and ISA. Notably, 18 clinical isolates (3.7%) from Indonesia
(n = 13), Thailand (n = 4) and China (n = 1) were resistant to 5FC
with MIC $32 mg/ml (Table S3). Most of 5FC resistant isolates
occurred in MC3 (n = 4), MC8 (n = 8) and MC17 (n = 5), and one
belonged to MC2 (Table S4). Approximately 33.3% of all MC17
isolates were resistant to 5FC and this was found to be highly
significant (p,0.0001, Chi-square; p = 0.0005, Fisher exact test)
when compared to the presence of 3.9 and 9.5% of resistant
isolates in MC8 and MC3, respectively. Ten FLU-resistant isolates
(2%) occurred in different countries, including China (n = 2), India
(n = 1), Indonesia (n = 5), and Thailand (n = 2) and belonged to
MC2 (n = 3), MC3 (n = 2), MC8 (n = 2), MC17 (n = 2) and one
isolate from India (number 25_17) that could not be assigned to
any MC (n = 1) (Table S4). Five isolates from Indonesia (strain
numbers 676, 1206, 1571, 3400, and 1048) that belonged to MC3
(n = 2), MC8 (n = 1) and MC17 (n = 2) were resistant to both 5FC
and FLU. Isolates obtained from HIV-negative patients and
HIVpositive patients did not differ in the susceptibility to three
antifungal compounds, including 5FC (p = 0.0298, t-test), FLU
(p = 0.0108, t-test) and VOR (p,0.0001, t-test). No significant
differences were observed for ITR (p = 0.0002, t-test), POS
(p,0.0001, t-test), and ISA (p,0.0001, t-test) when clinical and
environmental isolates were compared. When all isolates were
considered, the broadest ranges and highest MIC values were
those of 5FC (,0.063 to .64 mg/ml), followed by FLU (0.125 to
32 mg/ml). The lowest MIC range was observed for ISA (,0.016
to 0.125 mg/ml), followed by POS, VOR and ITR (,0.016 to
0.5 mg/ml) (Table 5). Among the azoles, FLU showed the
lowest activity (MIC90 = 4 mg/ml) and ISA the highest
(MIC90 = 0.063 mg/ml) (Table 5).
Microsatellites are an excellent tool to discriminate among C.
neoformans var. grubii isolates [17,33]. Indeed, 265 different
genotypes were observed in this Asian collection of 493 isolates.
Most isolates belonged to only eight microsatellite complexes
(MCs), including four new ones. Simpsons index of diversity,
however, showed that genetic diversity within each individual
population and each MCs was very high.
The predominant MC in each HIV status category is indicated in bold.
The genotypic structure of the C. neoformans var. grubii isolates
differed widely among the populations from the different
countries. This finding supports the hypothesis that local
geographic differences occur in Asia between C. neoformans var.
grubii populations that could be the result from different founder
effects and/or regional factors such as differences in the local
environment and climate . Recently, a population biology
study using MLST analysis of C. neoformans var. grubii compared
cryptococcal isolates from Thailand with a globally collected set of
isolates. This study showed that the Asian population was
genetically less diverse than those occurring in Africa and North
America . Furthermore, 15 of the 115 Chinese isolates (Table
S1) were investigated previously by M13 PCR fingerprinting and
identified as genotype VNIc . All these isolates clustered in
MC2, thus supporting the relatively limited genetic diversity
among Chinese cryptococcal strains as seen by AFLP. Thus, it
seems that the genetic diversity of cryptococcal isolates from Asian
countries is lower than that of populations occurring in other parts
of the world. The minimum spanning tree based on microsatellite
analysis showed that MC8 contained mostly Thai isolates
(Figure 1B), thus supporting the presence of limited genetic
diversity among this population as has been described before [36
39]. However, other Thai isolates from HIV-infected patients and
bird excreta from the North and Northeast occurred in MCs that
comprised also isolates from China and Japan (i.e., MC2 and
MC16). Can these findings be explained by assuming a relation
with bird migration, especially the East Asia-Australian flyway,
through which pathogenic microorganisms such as C. neoformans
var. grubii may disperse between China and Thailand ? The
scattered distribution of isolates from Kuwait and Qatar may be
due to migration of foreign workers from Southeast Asia who may
have carried isolates that were obtained from their country of
origin, similar as has been demonstrated for African immigrants in
France . Further support for this hypothesis is that young
children in USA acquired the organism from their surrounding
environments  and we think that this also happens elsewhere.
Isolates belonging to MC8 contained mainly cryptococcal
isolates from HIV-infected patients, whereas MC2 and MC16
comprised mostly isolates from non-HIV-infected patients
(Figure 1C). A recent report from Vietnam revealed two genotypic
Table 4. Distribution of C. neoformans isolates from clinical and environmental samples from Thailand and Japan in microsatellite
The predominant MCs in each sample type in these countries are indicated in bold.
Chiang Mai, Thailand
All C. neoformans isolates (n = 493)
Isolates from HIV-positive patients (n = 236)
Isolates from HIV-negative patients (n = 156)
Clinical isolates from Thailand and Japan (n = 68)
Environmental isolates from Thailand and Japan
(n = 67)
clusters based on AFLP analysis, viz., VNIc and VNId, that
contained isolates from HIV-negative and positive patients,
respectively . Thus genotypic differences were seen between
both patient categories based on AFLP analysis and this
observation is reminiscent to the differences that we observed in
the current microsatellite data. However, no comparison could be
made between the AFLP- and microsatellite typing because,
unfortunately, none of the Vietnamese isolates was available for
In Japan and Thailand, environmental isolates co-occurred with
clinical isolates in MC2, MC8, and MC16 (Figure 1A, C and D,
and Table 3), and these results suggest a genetic relatedness
between environmental and clinical cryptococcal isolates in these
countries. Such a relationship has been suggested before [34,43
46], but is at stake with a recent analysis from Cuba where the
clinical isolates largely belonged to a different MC than those
obtained from the environment . At present, the observed
differences between the genetic relationship of environmental and
clinical isolates in different parts of the globe are not easily
explained, but it may be that different environmental niches are
occupied in various locales.
Susceptibility analysis of 493 C. neoformans isolates to seven
antifungals yielded no change in MIC ranges and MIC50 and
MIC90 values of AMB and FLU when compared to previous
studies . The MIC ranges and values for ITR and the
three novel antifungal agents POS, ISA, and VOR were slightly
changed to those reported previously [22,4849]. The MIC50 and
MIC90 values for ISA were lower than those observed for the other
antifungal agents, thus corroborating previous observations
Importantly, resistance in approximately 4% of the isolates to
5FC was observed. Most 5FC-resistant isolates came from
Indonesia and Thailand where this drug is not in use .
In our study, we found that approximately 35% of all MC17
isolates were resistant to 5FC. These findings suggest that intrinsic
resistance to 5FC occurs among Southeast Asian isolates of C.
neoformans, especially in MC17 and in Indonesia. Intrinsic
resistance to 5FC in C. neoformans is uncommon and occurs with
a low incidence of approximately 12% as reported from the USA
in the 1990s . The reason for this relative high number of
5FC resistant isolates in the Southeast Asian region is not clear,
and needs further study. Furthermore, resistance to FLU was
observed. The FLU-resistant isolates occurred in different
countries, including China, India, Indonesia, and Thailand. C.
neoformans may develop resistance to FLU after treatment [52,54],
but the molecular origin of the resistance mechanism among the
Asian strains remains unknown.
Five out of 10 FLU resistant isolates (2 from MC3, 1 from MC8,
and 2 from MC17) from Indonesia were also not susceptible to
5FC. Our findings suggested that isolates in certain MCs,
especially MC17, may be prone to a decreased susceptibility to
antifungals, especially FLU and 5FC. The observed double
resistance to 5FC and FLU has not been reported before, and
may pose a risk for the patients infected with such isolates. Isolates
from HIV-infected and non-HIV-infected patients did not differ in
MIC values of 5FC, FLU, and VOR, but the MIC ranges of
isolates from HIV-infected patients were broader than those of
isolates from non-HIV-infected patients. The highest MIC values
of isolates from HIV-infected patients were 16 times higher for
5FC and FLU and two to four times as high as those from
nonHIV-infected patients, respectively.
This is the first extensive report on in vitro antifungal
susceptibility and genotyping of clinical and environmental isolates
of C. neoformans from several Asian countries. Based on the in vitro
susceptibility test results, the patients in the region seem to receive
appropriate antifungal therapy with AMB plus 5FC for induction
therapy followed by consolidation/maintenance therapy of FLU.
AMB is still the most effective agent to treat an infection with C.
neoformans . A Chinese study showed that the risk of death in
patients with cryptococcosis who did not receive AMB-based
initial therapy was about 79 times higher than those given AMB
. Another study from Thailand used different combinations of
antifungal therapies, i.e., AMB alone, AMB plus 5FC, AMB plus
FLU, or triple antifungals (AMB plus 5FC and FLU) to treat
cryptococcal meningitis showed that treatment of AMB combined
with 5FC remains a powerful treatment strategy that results in
rapid clearance of the pathogen . Our study did not reveal a
significant change in the MICs of AMB for C. neoformans, and
therefore, initial treatment with AMB remains the recommended
choice. However, given the well-tolerated nature and a excellent
activity against Cryptococcus strains, the new generation of triazoles
may become an important addition to the currently used
In summary, genotypic differences in microsatellite patterns
occur between C. neoformans populations from the Asian countries
studied. Most of the countries had a unique distribution of MCs
but the overall genetic diversity estimated by Simpsons index of
diversity was high. Good in vitro antifungal activity was observed,
but 5FC and FLU resistant, as well as 5FC/FLU double resistant
Table S2 Environmental Cryptococcus neoformans var.
grubii isolates from Thailand and Japan.
Table S4 The MIC range, MIC50, MIC90, and geometric
mean for the different microsatellite complexes (MCs)
of C. neoformans.
The opinions expressed by authors contributing to this journal do not
necessarily reflect the opinions of the Chinese Centers for Disease Control
and Prevention or the institutions with which the authors are affiliated. We
thank S.P. Simwami and M.C. Fisher for kindly donating cryptococcal
isolates and W.H.M. Chew for excellent technical assistance.
Conceived and designed the experiments: WP KK FH. Performed the
experiments: WP KK FH. Analyzed the data: WP KK FH TB JFM CHK.
Contributed reagents/materials/analysis tools: WP KK FH RW A.
Chakrabarti A. Chowdhary RI SJT ZK DI RS PS WL KC NI JWM
ICB TB JFM CHK. Wrote the paper: WP KK FH.
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