Geographically Structured Populations of Cryptococcus neoformans Variety grubii in Asia Correlate with HIV Status and Show a Clonal Population Structure
et al. (2013) Geographically Structured Populations of Cryptococcus neoformans Variety grubii in Asia
Correlate with HIV Status and Show a Clonal Population Structure. PLoS ONE 8(9): e72222. doi:10.1371/journal.pone.0072222
Geographically Structured Populations of Cryptococcus neoformans Variety grubii in Asia Correlate with HIV Status and Show a Clonal Population Structure
Matthew C. Fisher
Saad J. Taj-Aldeen
Leo J. J. van Iersel
Jacques F. Meis
Corne H. W. Klaassen
Oscar Zaragoza, Instituto de Salud Carlos III, Spain
Cryptococcosis is an important fungal disease in Asia with an estimated 140,000 new infections annually the majority of which occurs in patients suffering from HIV/AIDS. Cryptococcus neoformans variety grubii (serotype A) is the major causative agent of this disease. In the present study, multilocus sequence typing (MLST) using the ISHAM MLST consensus scheme for the C. neoformans/C. gattii species complex was used to analyse nucleotide polymorphisms among 476 isolates of this pathogen obtained from 8 Asian countries. Population genetic analysis showed that the Asian C. neoformans var. grubii population shows limited genetic diversity and demonstrates a largely clonal mode of reproduction when compared with the global MLST dataset. HIV-status, sequence types and geography were found to be confounded. However, a correlation between sequence types and isolates from HIV-negative patients was observed among the Asian isolates. Observations of high gene flow between the Middle Eastern and the Southeastern Asian populations suggest that immigrant workers in the Middle East were originally infected in Southeastern Asia.
Funding: This work was supported by a research grant from University of Phayao, Thailand (http://www.up.ac.th/). FH was funded by the Odo van Vloten
Foundation, the Netherlands. WH was granted by the National Natural Science Foundation of China (no. 31270180) and the National Basic Research Program of
China (no. 2013CB531606). MCF was granted by the Wellcome Trust (http://www.wellcome.ac.uk/) and the Biotechnology and Biological Sciences Research
Council, grant number BB/D52637X/1 (www.bbsrc.ac.uk). TB and SJT were supported by a grant from the Qatar National Research Fund, grant number NPRP
5298-3-086. 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
Indonesian Cryptococcus isolates. 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.
Cryptococcosis is one of the main fungal diseases in Asia due to
the AIDS pandemic and is caused by members of the Cryptococcus
neoformans/C. gattii species complex [1,2]. In South and Southeast
Asia, the number of HIV-infected patients that annually acquire
cryptococcosis is estimated to be over 140,000 , with the
majority of cases being caused by C. neoformans var. grubii .
The causative agent is an encapsulated opportunistic pathogenic
basidiomycetous yeast. Cryptococcosis caused by C. neoformans var.
grubii has also been reported to occur in immunocompetent
individuals in the Asian region, e.g. from China, Japan, Korea and
Taiwan [8,1316]. In Vietnam, cryptococcosis in both
immunocompromised and immunocompetent individuals was found to be
mainly caused by C. neoformans var. grubii . C. neoformans var.
grubii (serotype A) has a global distribution and is found in avian
excreta, especially from pigeons, and decaying wood . The
other variety, C. neoformans var. neoformans (serotype D), also has a
worldwide distribution but is more frequently encountered in
Europe [22,23]. C. gattii (serotypes B and C), a sibling species of C.
neoformans, is associated with many tree species in tropical and
subtropical regions [18,2427] and is a major cause of
cryptococcal meningitis in immunocompetent individuals. This latter species
has also been reported as a causative agent in
immunocompromised individuals, particularly HIV-infected patients and
solidorgan transplant patients [19,2831]. Since 1999, C. gattii emerged
in various outbreaks, e.g. at Vancouver Island (British Colombia,
Canada), the Pacific Northwest of the United States and more
recently in Mediterranean Europe [28,3235].
Several molecular typing methods, including
PCR-fingerprinting, randomly amplified polymorphic DNA (RAPD),
PCRrestriction fragment length polymorphism (PCR-RFLP), amplified
fragment length polymorphism (AFLP), microsatellite typing,
59 GTGAACAAGCTGCGGC 39
59 GGATTCAGTGTGGTGGAAGA 39
59 GGTTGTCAAGGTTGGAATCAACGG 39
59 GGAGCGGAAATGACGACCTTCTT 39
59 CAGACGACTTGAATGGGAACG 39
59 ATGCATAGAAAGCTGTTGG 39
59 GGCGATACTATTATCGTA 39
59 TTCTGGAGTGGCTAGAGC 39
59 CTTCAGGCGGAGAGAGGTTT 39
59 TTCAACCACGAATATGTA 39
59 AAGCCTCTCATCCATATCTT 39
59 ATGTCCTCCCAAGCCCTCGAC 39
59 TTAAGACCTCTGAACACCGTACTC 39
multilocus microsatellite typing (MLMT) and multilocus sequence
typing (MLST), have been developed for the investigation of the
epidemiology of species belonging to the C. neoformans/C. gattii
species complex [30,31,3641]. MLST is a typing system that has
several advantages over other commonly used typing methods,
because the technique is highly reproducible and MLST sequence
data can be stored in internet databases, such as http://www.mlst.
net/ and http://mlst.mycologylab.org. Thus, the data are portable
and exchangeable between laboratories. Recently, seven unlinked
genetic loci, i.e. CAP59, GPD1, IGS1, LAC1, PLB1, SOD1 and
URA5, that represent housekeeping genes, virulence factor coding
genes and the intergenic spacer of the ribosomal DNA have been
selected for MLST analysis of the C. neoformans/C. gattii complex by
the International Society of Human and Animal Mycoses
(ISHAM) working group on Genotyping of C. neoformans and C.
Previous studies that used MLST and AFLP to investigate the
population structure of C. neoformans var. grubii showed a
correlation between both methods and grouped the isolates into
three genetically different subgroups, named AFLP1/VNI,
AFLP1A/VNII/VNB and AFLP1B/VNII [36,39,42,43]. The
AFLP1/VNI and AFLP1B/VNII genotypes occur globally and
form a monophyletic cluster, whereas the AFLP1A/VNB
genotype occurs in Southern Africa, especially Botswana, but has also
been reported from Brazil [36,43]. Previously, recombination has
been observed within subpopulations in Botswana, but at the
global scale reproduction is mainly clonal . MLST has also
been used to trace the putative origin of Cryptococcus populations
[31,32,34]. Simwami and coworkers (2011) showed a correlation
between MLST types among Thai and African C. neoformans var.
grubii isolates that supported the hypothesis of long-distance
96uC 5min; 35 cycles: 96uC 30s,
56uC 30s, 72uC 1min;
96uC 5min; 35 cycles: 96uC 30s,
61uC 30s, 72uC 1min;
96uC 5min; 35 cycles: 96uC 30s,
61uC 30s, 72uC 1min;
96uC 5min; 35 cycles: 96uC 30s,
52uC 30s, 72uC 1min;
96uC 5min; 35 cycles: 96uC 30s,
56uC 30s, 72uC 1min;
96uC 5min; 35 cycles: 96uC 30s,
52uC 30s, 72uC 1min;
96uC 5min; 35 cycles: 96uC 30s,
63uC 30s, 72uC 1min;
Fraser et al., 2005
Meyer et al., 2003
dispersal from the African continent to Asia within the last
5,000 years .
In the current study, MLST was employed to determine the
genetic diversity and epidemiological relationships of a collection
of clinical and environmental C. neoformans var. grubii isolates that
originated from various geographic locations in Asia, including
countries from East, South/Southeast Asia and the Middle East.
We assessed the extent of recombination that occurs amongst
Asian C. neoformans var. grubii isolates. In addition, we determined
whether isolates from patients with a different HIV-status could be
distinguished using MLST. Finally, we analyzed whether
differences in susceptibility to various antifungal drugs correlated with
the observed MLST-based genotypic diversity.
Materials and Methods
Isolates and media
Three hundred and eleven isolates of Cryptococcus neoformans var.
grubii, including 244 clinical and 67 environmental isolates, were
obtained from the following sources: the Chinese Cryptococcus
Reference Centre at the Second Military Medical University,
Shanghai, China (nClinical = 86); the Department of Microbiology,
Meiji Pharmaceutical University, Tokyo, Japan (nClinical = 28;
nEnvironmental = 10); Prince of Wales Hospital, Hong Kong (nClinical
= 14); the Department of Parasitology, Faculty of Medicine,
University of Indonesia, Jakarta, Indonesia (nClinical = 40); the
Department of Medical Microbiology, Postgraduate Institute of
Medical Education and Research, Chandigarh, India (nClinical
= 61); the Department of Microbiology, Faculty of Medicine,
Health Sciences Centre, Kuwait University, Jabriya, Kuwait
(nClinical = 10) and the Mycology Unit, Microbiology Division,
Department of Laboratory Medicine and Pathology, Hamad
Medical Corporation, Doha, Qatar (nClinical = 5), and the
Department of Microbiology, Faculty of Medicine, Chiang Mai
University, Chiang Mai, Thailand (nEnvironmental = 57) (Table A
and B in Supplementary Tables S1). Furthermore, MLST data
obtained from 165 Thai clinical isolates included in a previous
study  complemented the strain set, resulting in a total of 476
isolates. The MLST profiles were compared to those included in
the global MLST dataset (http://mlst.mycologylab.org) and from
previous reports [16,44]. Isolate identification was done as
described by Pan et al., 2012 .
Mating- and serotype analysis by PCR
Extraction of genomic DNA was performed as previously
described . To determine mating- and serotypes, PCR
amplifications were applied as described previously [45,46]. C.
neoformans strains 125.91 (CBS 10512; aA; AFLP1/VNI), H99
(CBS 8710; aA; AFLP1/VNI), JEC20 (CBS 10511; aD; AFLP2/
VNIV), and JEC21 (CBS 10513; aD; AFLP2/VNIV) were used as
DNA from each isolate was amplified by PCR in 25 ml reaction
volumes for each of the seven MLST loci using the primers and
protocols described in Table 1. Each amplicon was subsequently
sequenced using the BigDye v3.1 Chemistry kit (Applied
Biosystems, Foster City, CA) using the same primers as used to
obtain the amplicons. Sequencing reaction products were purified
with Sephadex G-50 Superfine columns (Amersham Biosciences,
Piscataway, NJ) and a MultiScreen HV plate (Millipore, Billerica,
MA). An ABI 3700XL DNA analyzer (Applied Biosystems) was
used to determine the forward and reverse DNA sequences.
Consensus sequences were manually edited using SeqMan v8.0.2
(DNASTAR, Madison, WI) and were subsequently aligned with
MEGA v5.05 (www.megasoftware.net). Allele Types (ATs) were
assigned to each of the seven loci, resulting in a seven-digit allelic
profile for each isolate. The allelic profiles were then defined as
Sequence Types (STs) according to the ISHAM MLST consensus
scheme for C. neoformans/C. gattii species complex (http://mlst.
mycologylab.org). All sequences have been deposited in GenBank
under the accession number KC529683 to KC533008 (Table C in
Supplementary Tables S1) and novel ATs have been added to
http://mlst.mycologylab.org/. ATs analysed in Simwami et al.
(2011)  had the indels removed in order to make them
compatible for the then-current MLST dataset. However, the
current MLST scheme (http://mlst.mycologylab.org) includes
indels, and therefore we realigned the entire set of sequences
from the latter study in our analyses. This required the
reassignment of a number of ATs from the dataset of Simwami
et al. (2011)  (Table A in Supplementary Tables S1). For the
global comparison we used data from http://mlst.mycologylab.org
and recent reports by Cogliati and colleagues (2013)  and
Mihara and colleagues (2012) .
23 31 40 53 69 71 77 82 93 141 174 175 176 177 185 186 187 188 189 190 191 192 193 194 195
Sequence types (STs)
The predominant STs in each country are indicated in bold.
The determination of the extent of DNA polymorphisms, such
as haplotype diversity (Hd), nucleotide diversity (p), number of
polymorphic sites (S), average number of nucleotide differences (k)
and Wattersons estimate of the population scaled mutation rate
per sequence (hs), were calculated using DnaSP v5.10 (http://
www.ub.edu/dnasp/) . Tajimas D, Fu & Lis D*, Fu & Lis F*
and Fus Fs, tests for neutrality, were also calculated using DnaSP
Sequence types (STs)
23 31 40 53 69 71 77 82 93 141 174 175 176 177 185 186 187 188 189 190 191 192 193 194 195
The predominant STs in each HIV status category are indicated in bold.
v5.10. Negative values of these neutrality tests suggest evidence for
purifying selection or the excess of high-frequency variants,
whereas positive values suggest evidence for balancing or
overdominant selection or expansion of rare polymorphisms.
Genetic differentiation between populations was estimated using
Slatkin linearized FST statistics. Estimation of gene flow was
assessed using number of migrants per generation (Nm).
Investigation of population structure
The actual number of populations (K) among Asian C. neoformans
var. grubii in our study was estimated using Structure v2.3.4
(http://pritch.bsd.uchicago.edu/structure.html)  and
Structure Harvester (http://taylor0.biology.ucla.edu/
structureHarvester/) . Twenty-seven simulation runs were
conducted for each K from 2 to10 using a burn-in of 104
replications and 104 Markov Chain Monte Carlo (MCMC)
replications, respectively. The true K was calculated from the
average and standard deviation of each K using the ad hoc statistic
implemented in Structure Harvester. Graphic depictions of
population genetic structure were drawn from the coefficients of
the optimal K using CLUMPP v1.1.2 (http://www.stanford.edu/
group/rosenberglab/clumpp.html)  and Distruct v1.1 (http://
Two common statistics for multilocus linkage disequilibrium
analysis, the index of association (IA) and rBarD, were estimated
using Multilocus v1.3b (http://www.agapow.net/software/
multilocus/) . These statistics test the null hypothesis of free
recombination (i.e. no linkage disequilibrium). The observed
values of IA and rBarD were compared against the expected values
obtained with 1,000 randomized data sets. Using these criteria,
p,0.05 indicates that the null hypothesis of free recombination
should be rejected and, consequently, indicates the presence of
substantial clonal reproduction. In order to do so, the Pairwise
Homoplasy Index (PHI) test implemented in SplitsTree v4.0
(http://www.splitstree.org/)  and the pairwise linkage
disequilibrium analysis implemented in DnaSP v5.10 using Fishers
exact test were used to detect recombination events among
populations using separate alignments for all seven MLST loci. We
also used the reticulated network analysis using the CASS
algorithm  to detect recombination among the Asian
population using alignments of concatenated sequences for all
seven MLST loci. The genome of C. neoformans var. neoformans
strain B3501 ( = CBS 6900) was used as an outgroup for the CASS
The minimum spanning tree that represented the comparison
between the original sources of C. neoformans isolates and their
allelic profiles was generated by Phyloviz v1.0 using the
goeBURST algorithm (http://goeburst.phyloviz.net/) .
Phylogenetic analyses were performed using the
Neighborjoining method with 1,000 bootstrap replicates implemented in
MEGA v5.10. The substitution model of this analysis was the
uncorrected genetic distances (p-distance) model.
Antifungal susceptibility testing
The susceptibility pattern of seven antifungal drugs, namely
amphotericin B (AMB; Bristol Myers Squibb, Woerden, The
Netherlands), 5-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 tested for 14 clinical C.
neoformans var. grubii isolates from Hong Kong as described
previously . All other data were taken from the Pan et al.,
2012 study . Recently published epidemiological cutoff values
(ECVs) for C. neoformans var. grubii of AMB, 5FC, FLU, ITR, VOR
and POS were implemented in this study. The ECVs for C.
neoformans var. grubii of 5FC and FLU is 8 mg/ml, 0.25 mg/ml for
ITR, VOR and POS, and 1 mg/ml for amphotericin B,
Analysis of molecular variance (AMOVA), implemented in
Arlequin v188.8.131.52, was used to analyze the hierarchical structuring
of genetic variation among Asian C. neoformans populations using
the concatenated MLST sequences. Significance was assessed by
computing distance pairwise matrices from the MLST sequences
using 10,000 permutations .
Correlations between sequence types (STs) and origin of C.
neoformans isolates, including geographical origin and HIV-status of
patients, and antifungal drug susceptibility profiles (Pan et al.,
2012)  were determined using Chi-square or Fishers exact tests
and binary logistic regression (p,0.05). All statistical tests were
calculated using GraphPad Prism v5 for Windows (http://www.
graphpad.com/prism/prism.htm) (GraphPad Software, San
Diego CA) and XLSTAT v2013 (http://www.xlstat.com/)
The 476 C. neoformans var. grubii isolates in our dataset were
obtained from 228 HIV-positive patients, 134 HIV-negative
patients, and 47 from individuals with unknown HIV status
(Table A in Supplementary Tables S1), as well as 67 isolates from
avian droppings from Chiang Mai, Thailand and Tokyo, Japan
(Table B in Supplementary Tables S1). All isolates possessed
mating-type a and serotype A (i.e. were aA). The genetic diversity
of the 476 C. neoformans var. grubii isolates as assessed by MLST
revealed 28 sequence types (STs) (Table A, B and D in
Supplementary Tables S1), including 4 predominant STs, namely
ST4 (n = 105; 22.1%), ST5 (n = 156; 32.8%), ST6 (n = 96; 20.2%),
and ST93 (n = 52; 10.9%). ST31 and ST77 contained each 14
(2.9%) isolates. The remaining STs were confined to few isolates.
Most isolates from the Chinese, Hong Kong, and Japanese
populations belonged to ST5. Fourteen percent of the Thai
isolates (n = 30) belonged to this ST, which was here significantly
rarer than in China (Chi-square p,0.0001). ST4 and ST6 were
found to be the major MLST types in Thailand, while ST93 was
dominant in India and Indonesia (Chi-square p,0.0001). Fifteen
isolates from the Middle East were distributed among 10 STs
(Table 2). Most STs from this area consisted of a single isolate,
except ST5 and ST31. Most isolates of these latter two STs were
obtained from immigrant workers that originated from India,
Indonesia, Philippines, and Sudan (Table A in Supplementary
Among the 409 clinical isolates, 24 STs were identified and 16
of them contained clinical isolates only (Tables 34). STs 46 and
93 were the predominant STs and accounted for 83 (20.3%), 142
(34.7%), 78 (19.1%) and 52 (12.7%) isolates, respectively
(Chisquare p,0.0001). The remaining STs consisted of few isolates,
except ST31 and ST77 that consisted of 10 (2.4%) and 14 (3.4%)
isolates, respectively (Table 3). The majority of isolates from
HIVnegative patients belonged to ST5 (n = 92; 68.7%), while the
majority of isolates from HIV-positive people belonged to STs 4,
5, 6 and ST93 that accounted for 72 (31.6%), 27 (11.8%), 68
(29.8%) and 41 (18%) isolates, respectively (Table 3). We
investigated how genetic variation was structured across the Asian
clinical isolate dataset (i.e. isolates from HIV-positive, -negative
and unknown HIV-status patients) using AMOVA. This analysis
showed that allelic variation within populations (88.38%) was
higher than that observed among populations (11.62%)
(p,0.0001) (Table 4). When we compared clinical isolates from
HIV-positive patients categorized into three regions, East Asia,
Middle East and South/Southeast Asia, the variance within
populations was approximately 92% (p = 0.065460.0025),
indicating that significant variation in MLST genotypes occurred
among individuals within each regional population group. In
contrast, MLST genotypic variation within populations of isolates
from HIV-negative patients showed less genotypic differences
(36.80%) than the variance observed among populations (63.20%)
(p,0.0001). Chi-squared tests showed a relationship between HIV
status and STs (p,0.0001; Cramers V = 0.474) (Sheet S1). A
binary logistic regression test showed that ST5 is associated with
HIV status (p,0.0001) and the standardized (adjusted) Pearson
residuals showed that ST5 correlated to isolates obtained from
Sum of Squares
Variance components (%)
HIV-negative patients (Sheet S1). Of the 92 ST5 isolates, almost
all were sampled from East Asia, including China (n = 70; 76.1%)
and Japan (n = 20; 21.7%).
Only clinical and environmental isolates from Chiang Mai,
Thailand and Tokyo, Japan could be compared as no
environmental isolates could be studied from the other regions.
Sixtyseven environmental isolates from Chiang Mai, Thailand, and
Tokyo, Japan, belonged to 12 STs from which five STs (i.e.
ST141, ST176, ST188, ST190 and ST193) contained
environmental isolates only. STs 4, 5 and 6 were the predominant ST
types found among the environmental isolates (Chi-square
p,0.0001). The majority of environmental isolates from Chiang
Mai, Thailand, belonged to ST4 (n = 22; 38.6%) and 6 (n = 18;
31.6%) (Chi-square p = 0.0042), while almost all Japanese
environmental isolates belonged to ST5 (n = 8; 80%) (Fishers exact test
p = 0.064) (Table 5).
Association between sequence types and geographic
origin of Asian C. neoformans isolates
In order to determine the distribution of STs in different
geographical locations, minimum spanning trees and phylogenetic
analyses were undertaken based on allelic profiles using the
goeBURST algorithm and analysis of concatenated sequences
with the Neighbor-joining algorithm, respectively (Figure 1 and 2).
Three linages were observed in the minimum spanning tree.
Group 1 contained mostly isolates of STs 5, 186, 193 and 194 that
originated from China, Hong Kong, and Japan, and also
contained 30 out of 222 (13.6%) isolates from Thailand. Group
2 contained mostly isolates from Thailand (n = 184; 82.9%). The
predominant STs in this group were ST4 and 6. Group 3
comprised most of the Indian and Indonesian isolates that
belonged to STs 31, 77 and 93. (Figure 1A).
Phylogenetic analysis of the Asian isolates also showed three
clusters. Cluster I/VNI contained three major STs (ST4, 5 and 6)
that contained C. neoformans isolates from China, Hong Kong,
Japan, and Thailand. Most Indian and Indonesian isolates
occurred in cluster II/VNI, whereas cluster III/VNII contained
one ST (ST40) with only isolates from India (Figure 2). The
Middle East isolates showed a more scattered distribution
(Figure 1A and 2). Two isolates of ST31 came from Qatar, but
they were isolated from Indian and Sudanese immigrant workers
suggesting that their geographical origins lie elsewhere. Among the
clinical isolates, the minimum spanning tree and Maximum
Likelihood tree showed an association of the predominant STs,
including STs 4, 6 and 93, with C. neoformans isolates from
HIVpositive patients, while ST5, one of the predominant STs,
contributed mainly to isolates from HIV-negative patients
(Figure 1B and 2; Figure A in Supplementary Figures S1).
The global C. neoformans var. grubii MLST dataset that contained
179 isolates originating from Africa (n = 45), North/South
America (n = 31), Asia (n = 55) and Europe (n = 48) was compared
using the goeBURST algorithm with the isolates from Asia. Most
Asian C. neoformans isolates clustered together in one group, but a
few Asian isolates showed a scattered distribution. Two clusters of
African isolates and one cluster of European isolates were
observed. Some of isolates from those regions showed a scattered
distribution as did the North/South American isolates (Figure 1C).
Phylogenetic analysis using Neighbor-joining showed three clades
among the global C. neoformans var. grubii isolates. Clade I/VNII
contained isolates from Africa, North/South America, Asia and
Europe, clade II/VNB contained 17 STs from African isolates and
one ST comprising European isolates, and almost all Asian STs
occurred in clade I/VNI that also contained isolates from other
Nucleotide sequences of all seven loci studied (CAP59, GPD1,
IGS1, LAC1, SOD1, PLB1 and URA5) had between 6 and 15
polymorphic sites (Table 6). Locus IGS1 had the highest
nucleotide diversity (p) of 0.0045, followed by LAC1 (p = 0.0018)
and GPD1 (p = 0.0014). The average number of nucleotide
differences per sequence, i.e. the k-value, of most loci ranged
from 0.046 to 0.867, except for locus IGS1 that had a higher k
value of 3.274. Locus LAC1 showed the highest mutation rate
(hs = 2.226), while the other loci had low hs values ranging from
0.890 to 2.077. The number of haplotypes (alleles) at each locus
ranged from 3 for CAP59 and SOD1 to 7 for LAC1. Haplotype
diversity ranged from 0.013 for SOD1 to 0.658 for LAC1. The
neutrality tests, including Tajimas D, Fu & Li D*, Fu & Lis F*
and Fus Fs showed significant evidence of purifying selection for
all loci, except IGS1 that showed some evidence of balancing
selection (Table 6).
The number of polymorphisms of the concatenated sequences
of C. neoformans var. grubii isolates obtained from the East Asian
region, including China, Hong Kong and Japan, were lower than
those from South/Southeast Asian isolates (i.e. India, Indonesia
and Thailand), and those from the Middle East (i.e. Kuwait and
Qatar) (Figure 4A and Table E in Supplementary Tables S1). The
highest nucleotide diversity (p = 0.002), the highest average
number of nucleotide differences per sequence (k = 7.962), and
the highest haplotype diversity (Hd = 0.924) were found in C.
neoformans isolates from Kuwait and Qatar. C. neoformans isolates
from South/Southeast Asia had 75 polymorphic site (S) and 21
different haplotypes (h), and a high mutation rate per sequence
(hs = 11.816). Within the South/Southeast region, haplotype
diversity (Hd) of each population was almost similar, while other
nucleotide polymorphism estimation values of each population,
such as number of polymorphic sites (S), nucleotide diversity (p),
mutation rate (h) and the average number of nucleotide differences
per sequence (k), were different (Figure 4A, Table E in
Supplementary Tables S1). A significant signal of purifying
selection was observed in two C. neoformans populations, namely
the one from East Asia (i.e. the Japanese population) and the
South/Southeast Asian one (i.e. the Indian population), whereas
evidence of a balancing selection or expansion of rare
polymorphisms was found in the Indonesian population (Table E in
Supplementary Tables S1).
Compared to the global MLST dataset, the Asian population
had lower values of nucleotide diversity (p) and haplotype diversity
(Hd) of 0.0016 and 0.780, respectively, than those of the African
(p = 0.0062; Hd = 0.988), the North/South American (p = 0.0067;
Hd = 0.927) and European populations (p = 0.0025; Hd = 0.841)
(Figure 4B, Table F in Supplementary Tables S1). The Asian
population had lower numbers of polymorphic sites (S) and
number of haplotypes (h) of 81 and 32, respectively, than those
from the African populations (S = 124; h = 34), but they were
higher than those from the North/South American (S = 79; h = 16)
and European populations (S = 78; h = 17) (Figure 4B and Table F
in Supplementary Tables S1). Neutrality tests indicated that the
variation of all populations was neutral and population sizes did
not change. However, the North/South American population
showed evidence for a population overdominant selection,
whereas the remaining populations showed purifying selection or
Population structure of Asian C. neoformans var. grubii
Genetic differences and the level of gene flow between each of
two populations from the three Asian regions studied were
estimated using two statistics, FST and Nm, using concatenated
MLST sequences (4,022bp). Genetic differences of the East Asian
versus the South/Southeast Asian (FST = 0.351), and the East
Asian and Middle East (FST = 0.233) populations were higher than
those between the South/Southeast Asian and Middle East
populations (FST = 0.019) (Figure 4A, Table G in Supplementary
Tables S1). High levels of gene flow, indicated by an Nm value of
.1, were observed between the South/Southeast Asian
population when compared to the Middle East population (Nm = 25.76),
and, secondly, between the East Asian population and the Middle
East population (Nm = 1.64). However, the Nm value between the
South/Southeast Asian and the Middle East populations was
much higher than that between the East Asian and the Middle
East populations. When the MLST data of the Asian isolates were
compared to those from the African, North/South American, and
European continents, FST and Nm estimates were between 0.193
and 0.222, and between 1.76 and 2.09, respectively (Figure 4B,
Table H in Supplementary Tables S1), indicating the presence of
slight genetic differences, but the occurrence of significant gene
flow between the Asian population with those from Africa, North/
South America, and Europe.
Clusters of Asian C. neoformans var. grubii populations were
estimated using different numbers of populations that ranged from
K = 2 to K = 10 using Structure. The Evanno method implemented
in the Structure Harvester showed the highest delta K, an ad hoc
statistic, was produced at K = 3 (Figure BA in Supplementary
Figures S1). This implicates that K = 3 seems a good estimate for
the actual number of populations included in this study, thus
suggesting that three real genetic population clusters occur among
the Asian C. neoformans var. grubii isolates that do not fully
corroborate the geographically identified populations. The
distribution of these three populations differed between the countries
(Figure 5A). Almost all cryptococcal isolates from China, Hong
Kong and Japan, as well as some isolates from Thailand, belonged
to population I. The Thai and part of Indonesian isolates formed
Table 5. Distribution of sequence types (STs) of C. neoformans isolates from clinical and environmental samples from Thailand and
Chiang Mai, Thailand
Sequence types (STs)
The predominant STs in each sample type in these countries are indicated in bold.
population II. Indian isolates, together with part of the Indonesian
ones, formed the population III, whereas isolates from Kuwait and
Qatar belonged to diverse populations containing genotypes from
populations I-III (Figure 5A). Population structure analysis of the
global C. neoformans var. grubii isolates showed five genetic
populations (K = 5) (Figure BB in Supplementary Figures S1).
The population structure of the Asian isolates was the same as
described above, but two other major populations occurred,
namely an African and North/South American population, and
an European one. The African and American populations were
genetically diverse. Some isolates contained haplotypes occurring
among isolates from Asia and Europe and a few of the European
isolates shared haplotypes that occurred in isolates from other
continents (Figure 5B). However, whether these isolates represent
acquisitions from the local environment, or are due to the patient
traveling with an in situ latent infection is not known and requires
further sampling of environmental isolates.
The index of association (IA) and rBarD values were estimated
from the allelic data set to determine the presence of clonality and
recombination among Asian C. neoformans var. grubii populations.
For all isolates in the entire Asian population and in those from
each region (i.e. East Asia, the Middle East and South/Southeast
Asia) both IA and rBarD values strongly rejected the null
hypothesis of free recombination (Table 7). However, two
recombination events were observed using the pairwise linkage
disequilibrium routine implemented in DnaSp (Table I in
Supplementary Tables S1). One event occurred among locus
GPD1 and the remaining occurred among locus IGS1. Results of
the Pairwise Homoplasy Index (PHI) test (Table J in
Supplementary Tables S1) showed that no recombination occurred within
each locus across all Asian populations, however two
recombination events were observed among concatenated sequences of
isolates from East and South/Southeast Asia. CASS analysis
p-value rBarD p-value
IA: Index of Association.
rBarD: a modified statistics for multilocus linkage disequilibrium analysis.
(Figure C in Supplementary Figures S1) showed that no
recombination occurred among concatenated sequences of Asian
isolates. When allelic data that included the global MLST dataset
were included, rBarD showed that the overall population genetic
structure was in overall significant linkage disequilibrium. The
PHI test did not detect recombination events occurring within
each locus, but could detect the presence of recombination events
among concatenated sequence of the global MLST dataset
(Table J in Supplementary Tables S1).
Clinical isolates from Hong Kong China (n = 14)
All C. neoformans isolates (n = 476)
including those from Pan et al., 2012 
In vitro antifungal drug susceptibility values
The MIC values of 14 C. neoformans var. grubii isolates from
Hong Kong were determined for seven antifungal drugs, namely
AMB, 5FC, FLU, ITR, VOR, POS and ISA (Table 8) and all
were susceptible to all antifungal drugs tested. Due to the recent
introduction of epidemiological cutoff values, the overall results
slightly differ from those presented previously by Pan and
colleagues (2012). In this study, 21 clinical isolates (4.4%) from
Indonesia (n = 13), Thailand (n = 5), India (n = 2) and China (n = 1)
showed high MIC values $ 16 mg/ml of 5FC. Most of isolates
with high 5FC MICs occurred in ST4 (n = 3), ST5 (n = 2), ST77
(n = 2) and ST93 (n = 14) (Table A in Supplementary Tables S1).
Eight fluconazole (FLU)-resistant isolates (1.7%) occurred in India
(n = 1), Indonesia (n = 5), and Thailand (n = 2) and belonged to
ST5 (n = 1), ST6 (n = 1), ST93 (n = 5) and ST77 (n = 1) (Table A in
Supplementary Tables S1). All five 5FC and FLU resistant isolates
from Indonesia  belonged to ST93. One isolate from Thailand
(ST6) showed high MICs for FLU ($16 mg/ml), but also to VOR
($0.5 mg/ml) (Table A in Supplementary Tables S1).
Previous studies on the genetic structure of C. neoformans var. grubii
from Thailand using MLST data showed limited genetic variation
 with the majority of isolates belonging to STs 4, 5, and 6
(designated as ST44, 45 and 46, respectively, in the original paper by
Simwami et al., 2011, Table A in Supplementary Tables S1). Two of
these predominant STs (i.e. ST4 and ST6) differ only in four
nucleotides at a single locus . In the current study, we increased
the size of the Asian MLST dataset to include nearly 500 C.
neoformans var. grubii isolates originating from three broadly-defined
regions, namely East Asia (China, Hong Kong and Japan), South/
Southeast Asia (India, Indonesia and Thailand), and the Middle East
(Kuwait and Qatar). We found that 99.8% (n = 475) of these isolates
belonged to lineage VNI, 0.2% (n = 1) were VNII and 0% were
VNB. The C. neoformans var. grubii population from the East Asian
region showed less genotypic variation than those from South/
Southeast Asian and the Middle East regions, and most isolates
Table 8. The MIC range, MIC50, MIC90, and geometric mean for 14 Hong Kong and all 476 C. neoformans isolates for seven
belonged to ST5. This latter genotype was previously found to be the
main ST in China, Japan and South Korea [13,14,16] and was
reported previously as the MLST M5 genotype [13,14] (Note that
this ST was labelled as ST46 in Simwami et al., 2011  ). Our
data show that ST5 is the major MLST genotype among C.
neoformans var. grubii isolates in East Asia. At the global level, and in
agreement with previous findings , the Asian C. neoformans var.
grubii population was found to be less diverse than the African, the
North/South American, and the European populations. The
population genetic structure of the Asian population was found to
be different from the African, and the North/South American and
European populations, and contained very few isolates that shared
haplotypes occurring in these other populations. On the other hand,
the African, North/South American, and European populations
also contained some isolates that contained haplotypes occurring
among Asian isolates. These findings are in agreement with previous
investigations that showed a high genetic diversity of the African
population, especially genotype VNB, and less genetic diversity of
the Asian population. Note that the VNB lineage also contains also
Brazilian isolates [36,40,41,59,60], thus additional sampling at the
global scale may show a broader occurrence of this genotype.
In the current study, no evidence of recombination was detected
in the entire Asian region, nor in the smaller regions, or at the
global scale using the CASS -, Multilocus (rBarD) -, and PHI (in
case of separated loci) analyses. These results suggest that the
entire Asian C. neoformans var. grubii population is largely clonal as
was previously shown for the Thai population only . However,
the pairwise linkage disequilibrium analysis showed a
recombination event among sequences of GPD1 and IGS1 loci and the PHI
test also detected recombination events among concatenated
sequences in the East and South/Southeast Asia populations. This
may be due to non-meiotic reproduction as previously reported
[36,41,43] amongst isolates that are of the same a-mating type.
However, despite this limited recombination, clonal propagation
of genotypes predominated leading to a widespread occurrence
and overrepresentation of clonal genotypes as has also been seen in
several other pathogens, such as Neisseria meningitides, Mycobacterium
tuberculosis, Fusarium oxysporum, and Leishmania tropica .
Our analyses revealed a significant association between
predominant sequence types (STs) and their geographical origin in Asia that
was not encountered before. These local geographic differences
could result from different founder effects and/or regional factors,
and may be due to environmental and climate differences
[6,8,62,63]. Sequence type ST5 was the predominant MLST
genotype found in East Asia and the North of Thailand. Due to the
association of C. neoformans with birds, dispersal may have occurred
via the East Asian-Australian flyway [8,64], and may contribute to
the broader distribution of these genotypes. As most bird migrations
generally happen twice a year depending on weather conditions, this
may also contribute to the observed limited dispersal and low gene
flow estimates between East Asia and Northern Thailand. Thus, it
seems that Asian C. neoformans isolates efficiently reproduce clonally,
and that rare recombination events may result in an increased
genetic variation at some locales, e.g. due to same-sex mating or rare
MATa x MATa crossings .
A scattered MLST distribution of the Middle East population was
observed, similar to previous findings using microsatellite analysis
. Owing to a low number of isolates (n = 15) with ten haplotypes,
the Middle East C. neoformans var. grubii population showed the
highest haplotype diversity in our dataset. Seven out of these 15 C.
neoformans var. grubii isolates were obtained from immigrant workers,
mainly originating from South/Southeast Asia, who may have
acquired the yeast in their home countries and, subsequently,
carried the pathogen when moving to the Middle East region.
Cryptococcal species are known to have a high prevalence of
subclinical infection due to infection in childhood . Immigrants
and tourists in Europe showed the same phenomenon of being
infected by strains that were obtained from their home-country
[31,46,67]. The observed high level of gene flow between the
Middle East and the South/Southeast Asian populations supports
this human migration hypothesis. Minimum spanning tree analysis
showed that most isolates from India and Indonesia belonged to the
same STs (i.e., ST77 and ST93) and a high level of gene flow was
observed between these two populations. This observation,
unfortunately, is not easy to explain and requires more sampling
especially from the environment in these regions.
HIV-status, STs and geographic origins were found to be
confounded. However, a significant correlation occurred between
the predominant ST5 and HIV-negative patients in Asia. Thus,
our study reinforced that genetic differences occur between C.
neoformans var. grubii isolates from HIV-positive and HIV-negative
patients in Asia [8,17,68,69]. Therefore, this finding may explain
the observed correlation of MLST genotypes and HIV status in
our study. To unravel the effect of geographically determined
genetic variation in the pathogen and its link to HIV status,
extensive sampling of environmental isolates is needed across the
region in order to decouple the HIV-status of individuals from the
geographical origin of the isolates. Next to possible genetically
determined differences amongst Cryptococcus isolates, human
factors, such as anti-interferon-c and
anti-granulocyte-macrophage colony-stimulating factor autoantibodies that have been
observed in Taiwanese and Thai patients to be associated with
adult-onset immunodeficiency without HIV-infection , may
contribute to the observed specific pathogen-host correlations.
Recently, epidemiological cutoff values (ECVs) have been
defined for the major antifungals against C. neoformans and C. gattii
[56,57]. When we used those values, the interpretation of the
overall susceptibility results of all isolates, including those
presented by Pan et al. , differed slightly from the interpretation
given by these last authors. Twenty-four clinical isolates mainly
from Indonesia and Thailand showed high MIC values of 5FC,
FLU and VOR. As 5FC is not used in those two countries,
resistance to this compound is unlikely to be induced by patient
treatment and the origin of this resistance needs further studies.
MLST typing showed significant genotypic variation between C.
neoformans var. grubii populations originating from different Asian
regions. Each country had an unique distribution of STs,
especially of the predominant STs. Overall, the Asian population
showed limited genetic diversity and reproduction is mainly clonal
when compared with data from the global C. neoformans var. grubii
MLST dataset. A correlation between STs and HIV-negative
status, and resistance traits was observed. A largely clonal
reproduction strategy helps to maintain these regional differences
that are clinically relevant due to their association with the
HIVstatus of the patients that also differs between the regions studied.
Sheet S1 Chi-square test of Asian C. neoformans
according to sequence types (STs) and HIV status.
Conceived and designed the experiments: TB KK FH JFM. Performed the
experiments: KK FH WP SS MF WM LT JFM CHWK TB. Analyzed the
data: KK FH WP SS MF WM LT LJJVI JFM CHWK TB. Contributed
reagents/materials/analysis tools: KK FH WP SS MF RW A. Chakrabarti
A. Chowdhary RI SJT ZK MI DI RS PS WL KC VV WM LT LJJVI JFM
CHWK TB. Wrote the paper: KK FH WP MF WM JFM TB.
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