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mycoses Diagnosis,Therapy and Prophylaxis of Fungal Diseases

Original article

Molecular epidemiology and in vitro antifungal susceptibility testing


of 108 clinical Cryptococcus neoformans sensu lato and Cryptococcus
gattii sensu lato isolates from Denmark

Ferry Hagen,1 Rasmus Hare Jensen,2 Jacques F. Meis1,3 and Maiken Cavling Arendrup2
1
Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands, 2Department of Microbiological
Surveillance and Research, Unit of Mycology, Statens Serum Institut, Copenhagen, Denmark and 3Department of Medical Microbiology, Radboudumc,
Nijmegen, The Netherlands

Summary Cryptococcosis is mainly caused by members of the Cryptococcus gattii/Cryptococcus


neoformans species complexes. Here, we report the molecular characterisation and
in vitro antifungal susceptibility of Danish clinical cryptococcal isolates. Species, geno-
type, serotype and mating type were determined by amplified fragment length poly-
morphism (AFLP) fingerprinting and qPCR. EUCAST E.Def 7.2 MICs were determined
for amphotericin B, flucytosine, fluconazole, voriconazole and isavuconazole. Most iso-
lates were C. neoformans (serotype A; n = 66) and belonged to genotype AFLP1/VNI
(n = 61) or AFLP1B/VNII (n = 5) followed by Cryptococcus deneoformans (serotype D;
genotype AFLP2, n = 20), C. neoformans 9 C. deneoformans hybrids (serotype AD;
genotype AFLP3, n = 13) and Cryptococcus curvatus (n = 2). Six isolates were C. gattii
sensu lato, and one isolate was a C. deneoformans 9 C. gattii hybrid (genotype AFLP8).
All isolates were amphotericin B susceptible. Flucytosine susceptibility was uniform
MIC50 of 48 mg l 1 except for C. curvatus (MICs >32 mg l 1). Cryptococcus gattii
sensu lato isolates were somewhat less susceptible to the azoles. MICs of fluconazole
(>32 mg l 1), voriconazole (0.5 mg l 1) and isavuconazole (0.06 and 0.25 mg l 1
respectively) were elevated compared to the wild-type population for 1/19 C. deneofor-
mans and 1/2 C. curvatus isolates. Flucytosine MIC was elevated for 1/61 C. neofor-
mans (>32 mg l 1). Antifungal susceptibility revealed species-specific differential
susceptibility, but suggested acquired resistance was an infrequent phenomenon.

Key words: Cryptococcus neoformans, Cryptococcus gattii, Cryptococcus deneoformans, Cryptococcus bacillisporus,
Cryptococcus deuterogattii, antifungal susceptibility testing, amplified fragment length polymorphism fingerprinting,
multilocus sequence typing.

thoroughly taxonomically revised.1 The majority of


Introduction
cryptococcosis cases are caused by members of the
Cryptococcosis is the encompassing description of Cryptococcus gattii and Cryptococcus neoformans species
disease caused by species within the basidiomycetous complexes, while infections with other species have
yeast genus Cryptococcus that recently has been rarely been reported till date.24 Cryptococcosis is
mainly diagnosed among individuals who have an
Correspondence: Dr Maiken Cavling Arendrup, Department of Microbio- impaired immune system, and it has been estimated
logical Surveillance and Research, Unit of Mycology, Statens Serum Institut, that annually nearly a million HIV-infected patients
Artillerivej 5, 43/317, DK-2300 Copenhagen S, Denmark.
Tel.: +45 3268 3223. Fax: +45 3268 3868.
develop cryptococcal meningitis with 625 000 deaths.5
E-mail: maca@ssi.dk Cryptococcus cells can remain dormant for decades after
they have been inhaled as basidiospores or desiccated
Submitted for publication 11 February 2016 yeast cells, and can be activated when the host immune
Revised 29 February 2016 status is attenuated.68
Accepted for publication 13 March 2016

2016 Blackwell Verlag GmbH doi:10.1111/myc.12507


F. Hagen et al.

Molecular techniques, such as PCR fingerprinting by extremely rare C. gattii s.s. with either C. deneoformans
M13 and (GACA)4 primers, restriction fragment length (serotype BD, genotype AFLP8) or C. neoformans (sero-
polymorphism (RFLP) analysis of the PLB1 and URA5 type AB, genotype AFLP9) were firstly described nearly
gene, amplified fragment length polymorphism (AFLP) a decade ago (Fig. 1).13,14
fingerprinting, microsatellite typing and multilocus The epidemiology of Cryptococcus infections in Eur-
sequence typing (MLST), have been regarded as help- ope has been studied, as is the case for many other
ful tools in unravelling the genotypic diversity.912 geographical regions.15,16 However, the majority of
The taxonomy of the C. gattii and C. neoformans spe- recent European epidemiological studies that made
cies complexes has been revised due to the differences use of molecular typing techniques came from Wes-
between both species in terms of genetics, physiology, tern and Mediterranean Europe.15 For the Scandina-
pathogenicity and ecology (Fig. 1).6,9,10,12 The two vian countries of Denmark, Finland, Iceland, Norway
varieties of C. neoformans were raised to the species and Sweden, recent epidemiological data are
level as C. neoformans (formerly var. grubii) represent- sparse.1719 To update the current knowledge about
ing serotype A isolates with genotypes AFLP1/VNI, cryptococcal infections in Scandinavia, in particular
AFLP1A/VNB/VNII and AFLP1B/VNII, and Cryptococ- for Denmark, a set of Cryptococcus isolates was molec-
cus deneoformans (formerly var. neoformans) represent- ularly investigated by mating and serotyping, AFLP
ing serotype D and genotype AFLP2/VNIV isolates and MLST genotyping, and by antifungal susceptibility
(Fig. 1).10 Cryptococcus gattii was separated into five testing according to the EUCAST E.Def 7.2 reference
species, Cryptococcus gattii sensu stricto (serotype B, method.
genotype AFLP4/VGI), Cryptococcus bacillisporus (sero-
type C, genotype AFLP5/VGIII), Cryptococcus deutero-
Materials and methods
gattii (serotype B, genotype AFLP6/VGII), Cryptococcus
tetragattii (serotype C, genotype AFLP7/VGIV) and
Isolates and media
Cryptococcus decagattii (serotype B, genotype AFLP10)
(Fig. 1).10 Hybrids of C. neoformans 9 C. deneoformans The mycological culture collection of the Staten Serum
(serotype AD, genotype AFLP3/VNIII), and although Institute contained 114 cryptococcal isolates, which

Figure 1 Cryptococcal species identification, sero- and genotypes according to former and the current Cryptococcus taxonomy.1,10

2 2016 Blackwell Verlag GmbH


Cryptococcosis in Denmark during 19732013

were preserved during the period 19732013 and


Mating-type and serotype determination
stored in glycerol at 80 C. One hundred and eight
isolates were viable and included in the current study. The mating-type and serotype of C. neoformans was
All isolates were previously identified by conventional determined using a recently established real-time
mycology procedures, including API ID32C (Biomer- PCR method that targets the mating-type a and a
ieux, Marcy lEtoile, France), as being members of the allele of the RUM1 gene for either the serotype A or
C. gattii species complex (n = 7; 6.5%), C. neoformans D background.20 Genomic DNA of the reference
species complex (n = 99; 91.7%) and Cryptococcus strains CBS10512 (=125.91; aA; genotype AFLP1/
curvatus (n = 2; 1.9%). Isolates were cultured onto VNI), CBS10511 (=JEC20; aD; genotype AFLP2/VNIV),
Sabouraud dextrose agar (Oxoid, Basingstoke, UK) for CBS8710 (=H99 = CBS10515; aA; genotype AFLP1/
48 h at 30 C. VNI) and CBS10513 (=JEC21; aD; genotype AFLP2/
VNIV) were included as controls.
A third recently described real-time PCR assay that
Antifungal susceptibility testing
targets the multicopy intergenic spacer region (IGS1)
The determination of broth dilution minimum inhibi- was applied to detect the presence of C. gattii sensu lato
tory concentrations of antifungal agents was carried among the samples.21 The reference strain CBS6956
out according to EUCAST guidelines (E.Def 7.2) for (aB; genotype AFLP6/VGII) was included as a control.
amphotericin B, flucytosine, fluconazole, voriconazole The mating-type of C. gattii sensu lato isolates were
and isavuconazole.2 Manufacturers and stock solutions determined, as described before, by a conventional
(5000 mg l 1) in water (flucytosine) or DMSO (the PCR method that targets either the mating-type a or a
remaining compounds, dimethyl sulfoxide (DMSO, allele of the STE12 gene.23 As a control, the reference
D8779, Sigma-Aldrich, Vallensbk Strand, Denmark) isolates CBS6955 (aB; genotype AFLP5/VGIII) and
were the following: amphotericin B, flucytosine and CBS6956 (aB; genotype AFLP6/VGII) were included.
fluconazole (Sigma-Aldrich), isavuconazole (Astellas Agarose gel electrophoresis was carried out with 5 ll
Pharma Europe, Middlesex, UK) and voriconazole (Pfi- PCR product to check the presence of a fragment.
zer, Ballerup, Denmark). The drug concentration range
studied was 0.034 mg l 1 for amphotericin B, isavu-
Amplified fragment length polymorphism
conazole and voriconazole, and 0.2532 mg l 1 for
fingerprinting
5-flucytosine and fluconazole. Plates were incubated at
30 C to promote growth as suggested in EUCAST All isolates were subjected to amplified fragment
E.Def 7.2 and read when the growth was 0.200 OD length polymorphism (AFLP) genotyping following the
above the background level. Candida parapsilosis ATCC method as described before.22,23 Fragments were anal-
22019 and Candida krusei ATCC 6258 were included ysed on an ABI3500xL Genetic Analyzer (Applied
as quality controls. Biosystems, Foster City, CA, USA); for this purpose,
the amplicons were 209 diluted with ddH2O, and 1 ll
of this dilution was added to 0.1 ll LIZ600 internal
Genomic DNA extraction
size marker (Promega, Leiden, The Netherlands) and
A loop full of cryptococcal cells from 48 h old cul- 8.9 ll ddH2O. Raw data was further processed by
tures were suspended in 400 ll Bacterial Lysis Buffer using Bionumerics v6.6 (Applied Maths, Sint-Martens-
(Roche Diagnostics, Almere, The Netherlands) fol- Latem, Belgium) and a dendrogram was generated
lowed by bead beating the cell suspension by using based on the UPGMA algorithm.
green cap tubes prefilled with 1.4 mm zirconium
beads in the MagNA Lyser (both Roche Diagnostics).
Multilocus sequence typing of C. gattii sensu lato
The lysed cell suspension was subsequently inacti-
isolates
vated for 15 min at 95 C. Genomic DNA extraction
was automatically carried out with the Pathogen To link C. gattii sensu lato isolates from the current
Detection 200 protocol with a MagNA Pure 96 robot study with those from a large collection of genotyped
(Roche Diagnostics) for which an input of 200 ll environmental, veterinary and clinical isolates, multi-
lysed cell suspension was used, purified DNA was locus sequence typing (MLST) was performed.11 The
finally eluted in 100 ll elution buffer (Roche Diagnos- nuclear loci CAP59, GPD1, IGS1, LAC1, PLB1, SOD1
tics). The DNA samples were stored at 20 C until and URA5 were amplified and sequenced as described
further use. before.6,11 Obtained sequences were concatenated and

2016 Blackwell Verlag GmbH 3


F. Hagen et al.

compared to available sequences.6,2430 Phylogenetic obtained ITS sequences with that of the recently estab-
analysis on the concatenated dataset was carried out lished ISHAM-ITS database showed that both Danish
using the software package MEGA v6.6.31 A maximum isolates were 100% identical to that of the reference
likelihood analysis was performed on all aforemen- sequence for C. curvatus. The taxonomy of the poly-
tioned available MLST data, based on that a subtree phyletic genus Cryptococcus has recently been thor-
was generated that included isolates genetically closely oughly revised; as a consequence, Cryptococcus
related to the Danish C. gattii sensu lato. Subsequently, curvatus was transferred to the novel genus Cutaneotri-
a 10009 bootstrap maximum likelihood analysis was chosporon and retained is epithet curvatus.1
initiated with the best fitting nucleotide substitution
model for the Cryptococcus MLST dataset that was pre-
Multilocus sequence typing of C. gattii sensu lato
viously observed to be HKY with models incorporating
isolates
invariant sites (+I) along with discrete gamma rate
categories (+G).6,31 Obtained sequences were deposited The six isolates that were identified by AFLP genotyp-
in Genbank under accession numbers KM230242 ing as being C. gattii sensu lato were subjected to
KM230318. MLST to determine their genetic relatedness to a glo-
bal set of isolates (Fig. 2). The two C. gattii sensu
stricto isolates DK0821 and DK0842 were found to
Results
be genetically indistinguishable from isolates
ATCC64062, CBS1622, CBS5757 and CBS6992
Mating-, sero- and genotyping of C. gattii/
(Fig. 2). Two of the three C. bacillisporus isolates were
C. neoformans species complex isolates
found to be genetically closely related (DK0841 and
The majority of the isolates were C. neoformans repre- DK0894) to each other as well as to a cluster of
sented by the genotypes AFLP1/VNI (n = 61; 56.5%) CN043, CBS7819, CBS6993, CBS5758, 7597057,
and AFLP1B (n = 5; 4.6%) (Fig. 1). C. deneoformans 384C, IHEM10079, B9422 and B8260 (Fig. 2). Cryp-
genotype AFLP2/VNIV (n = 20; 18.5%) was the second tococcus bacillisporus isolate DK0893 was found to be
most common followed by the C. neoformans 9 C. dene- similar to CBS6996 and ATCC24065, an environmen-
oformans serotype AD hybrid genotype AFLP3/VNIII tal and clinical isolate, respectively, from the USA
(n = 13; 12.0%) (Fig. 1). Cryptococcus gattii sensu lato (Fig. 2). The single C. deuterogattii isolate DK0022
was found to be represented by seven isolates dis- clustered together with isolates that had a South
tributed over the three species C. gattii sensu stricto American origin (Fig. 2).
AFLP4/VGI (n = 2; 1.9%), C. bacillisporus AFLP5/VGIII
(n = 3; 2.8%) and C. deuterogattii AFLP6/VGII (n = 1;
Antifungal susceptibility testing according the EUCAST
0.9%) (Fig. 1). One isolate (0.9%) was an interspecies
method
hybrid between C. gattii sensu stricto and C. deneofor-
mans, known as genotype AFLP8 with serotype BD. All isolates were susceptible to amphotericin B defined
All 66 C. neoformans isolates were found to be aA. Of as MICs being 1 mg l 1 with only discrete differ-
the 20 C. deneoformans isolates, eight were found to be ences among the species/varieties (Table 1, Fig. 3).
aD and 12 aD. Of the 13 C. neoformans 9 C. deneofor- Thus, C. neoformans was slightly less susceptible than
mans hybrids, five were found to be aD-aA, one was aA- C. deneoformans (MIC50 0.125 vs. 0.06 mg l 1, and
aD. For seven isolates, only the serotype D background GM 0.1490.018 vs. 0.096 mg l 1, respectively,
could be detected with one isolate being aD and the Table 1). Similarly, the in vitro activity of flucytosine
remaining six being aD. All the six C. gattii sensu lato was uniform with an MIC50 of 48 mg l 1 for all spe-
isolates were found to be mating-type a, and the mating- cies with the exception of the two C. curvatus isolates
types of the interspecies hybrid C. gattii sensu that were both resistant (MICs of >32 mg l 1).
stricto 9 C. deneoformans isolate could not be determined. However, flucytosine MIC was clearly elevated for
1/61 C. neoformans AFLP1/VNI isolate (>32 mg l 1,
Fig. 3) and a tail of C. deneoformans and C. neofor-
Identification of Cryptococcus species other than
mans 9 C. deneoformans hybrid isolates with MICs in
C. gattii/C. neoformans species complex
the 3264 mg l 1 range were observed.
The two isolates previously identified as C. curvatus by The C. gattii sensu lato isolates were less susceptible
conventional phenotypic identification were confirmed to the three azoles and to fluconazole in particular com-
by sequencing of the ITS region.32 Comparing the pared to the other species (Fig. 3). Thus, fluconazole,

4 2016 Blackwell Verlag GmbH


Cryptococcosis in Denmark during 19732013

Figure 2 Maximum likelihood 10009


bootstrapped phylogenetic analysis based
on the ISHAM consensus MLST scheme11
of Danish C. gattii sensu lato isolates
(in bold) compared to a set of globally
collected isolates.6,2729 Numbers next to
branches represent bootstrap values, only
those >80 are show.

voriconazole and isavuconazole MIC50s were 16, 0.25 (Table 1, Fig. 2). Finally, fluconazole (>32 mg l 1),
and 0.06 mg l 1 for C. gattii sensu lato in contrast to voriconazole (0.5 mg l 1) and isavuconazole (0.06
24, 0.06 and <0.030.06 mg l 1 for the others and and 0.25 mg l 1 respectively) were elevated for 1/19
geometric mean MICs followed the same pattern C. deneoformans and 1/2 C. curvatus isolates (Fig. 2).

2016 Blackwell Verlag GmbH 5


F. Hagen et al.

Discussion

2 to >32

1 to >32

1 to >32
Range

116
>32
Amplified fragment length polymorphism fingerprint-
Table 1 Amphotericin B, voriconazole, isavuconazole, fluconazole and flucytosine MICs against 108 Danish Cryptococcus isolates. Susceptibility testing was done following the

4
ing showed that the majority of the Danish cryptococ-

8.80
4.00

6.00
12.62

14.25
GM cal isolates (n = 61; 56.5%) represented C. neoformans,
Flucytosine

56 isolates (51.9%) belonged to the major genotype


AFLP1/VNI and a small proportion (n = 5; 4.6%)
MIC50

belonged to the genotype AFLP1B/VNII. The large pro-


4

8
4
4
4
portion of C. neoformans isolates mirrors the observa-
0.5 to >32

0.5 to >32

2 to >32
tions from other European epidemiological studies.

832
Range

0.58

48
Isolates collected during the period 19772007 in the

4
Netherlands were found to be represented by 79.3%
(n = 238) of genotype AFLP1/VNI and 2.3% (n = 7)
2.96

8.96
6.40
4.93
21.33
GM

of genotype AFLP1B/VNII.33 A retrospective study


Fluconazole

from Germany identified 78 C. neoformans isolates


MIC50

(77.2%) during the period 20042010.34 A prospec-


4

4
8
2
16

tive multicentre study from France reported that the


<0.030.06

<0.030.25
<0.030.06
<0.030.06

0.060.25

majority of the isolates during the period 19972001


<0.032

was C. neoformans (n = 161; 72.5%); of note, only


Range

<0.03

C. neoformans and C. deneoformans infections were


identified in this study.35 An earlier report from
0.039

0.065
0.042
0.041
0.400
0.155
Isavuconazole

France conducted from 1985 to 1993 included 1057


GM

cases of cryptococcosis, but only 413 isolates were


MIC50

<0.03

0.06
<0.03
<0.03
0.06

available for serotyping and the majority of them were


C. neoformans (n = 326; 78.9%).36 However, in
Mediterranean Europe, the number of C. neoformans
<0.030.25

<0.030.5

<0.030.5
0.060.5
0.1251

isolates in epidemiological surveys is often lower. For


Range

example, in Serbia, 58.8% (n = 20) of the studied iso-


0.06

0.06

lates were genotype AFLP1/VNI and 2.9% (n = 1)


genotype AFLP1B/VNII,20, while a report from a single
0.102

0.104
0.060
0.081
0.281
0.563
Voriconazole

GM

institute in Spain reported 44.8% (n = 26) C. neofor-


mans infections.37 The Spanish study reported 19%
MIC50

(n = 11) and 36.2% (n = 21) of the isolates being


0.06

0.06
0.06
0.06
0.25

C. deneoformans (genotype AFLP2/VNIV) and hybrids


between C. neoformans 9 C. deneoformans (genotype
EUCAST E.Def 7.2 method using incubation at 30 C for 48 h.

<0.030.25

0.060.25
<0.030.25
0.060.25
0.060.25
<0.031

AFLP3/VNIII) respectively. In Serbia, 10 isolates


0.125
Range

(29.4%) were found to be C. deneoformans and three


(8.8%) were hybrid serotype AD isolates.20 The per-
Amphotericin B

0.112

0.180
0.149
0.096
0.123
0.155

centage of C. deneoformans isolates in the current Dan-


GM

ish epidemiological study equals that of the Spanish


study (n = 20; 18.5% vs. n = 11; 19%).37 The neigh-
MIC50

0.125
0.125
0.06

0.06
0.06

bouring countries Germany and the Netherlands


reported a lower incidence of C. deneoformans infec-
C. deneoformans, AFLP2/VNIV (20)

tions (n = 15; 14.9% vs. n = 36; 12.0% respectively)


Cryptococcus gattii sensu lato (6)
C. neoformans, AFLP1B/VNII (5)
C. neoformans, AFLP1/VNI (61)

when compared to Denmark.33,34 The number of clini-


neoformans 9 Cryptococcus

cal C. neoformans 9 C. deneoformans hybrids in Den-


Cryptococcus curvatus (2)
Cryptococcus interspecies
deneoformans hybrid,

mark is low (n = 13; 12.0%), but, again, a lower


(number of isolates)

incidence was reported from Germany (n = 8; 7.9%)


Species, AFLP type

hybrid, AFLP8 (1)


AFLP3/VNIII (13)

and the Netherlands (n = 14; 4.7%).33,34


Cryptococcus

Seven Danish isolates were found to be C. gattii


sensu lato by phenotypic characterisation, and subse-
quent AFLP genotyping confirmed that six belonged

6 2016 Blackwell Verlag GmbH


Cryptococcosis in Denmark during 19732013

Figure 3 MIC data for the Danish


Cryptococcus isolates by species. Species
isolates with intrinsic reduced susceptibil-
ity compared to that for C. neoformans
are highlighted with arrows. Isolates with
potential acquired resistance are
indicated by circles.

indeed to this species complex, while one isolate was The six C. gattii sensu lato isolates were further
found to be a rarely reported interspecies hybrid characterised by MLST analysis, and the two C. gattii
between C. gattii sensu stricto (genotype AFLP4/VGI) sensu stricto isolates DK0821 and DK0842 (genotype
and C. deneoformans (genotype AFLP2). So far, world- AFLP4/VGI) were genetically indistinguishable from
wide, only a few C. gattii sensu stricto 9 C. neoformans two North American isolates, as well as from the type-
interspecies hybrids with genotype AFLP8 have been strain CBS1622 which had been isolated in France
described and in-depth genetic analyses showed that during the early 20th century (Fig. 2).6,10 Interest-
these interspecies hybrids, next to those that exhibit ingly, the closest relatives of this cluster originated
genotype AFLP9, had mixed phenotypic characteristics from the South African environment, suggesting that
from both parental species and that the hybrid gen- the closely related clinical isolates from Denmark,
ome had loss part of its genetic material.10,13,14 France and the USA somehow had a link with the

2016 Blackwell Verlag GmbH 7


F. Hagen et al.

African continent (Fig. 2). The three isolates DK0841, and amphotericin B (0.5 and 1 mg l 1 respectively),
DK0893 and DK0894 were molecularly characterised flucytosine (1 and 8 mg l 1 respectively), fluconazole
as being C. bacillisporus (genotype AFLP5/VGIII), but (4 and 8 mg l 1 respectively), voriconazole (0.12 and
could be distinguished from each other by MLST 0.5 mg l 1 respectively) and isavuconazole (0.03 and
(Fig. 2). Nevertheless, all Danish C. bacillisporus iso- 0.25 mg l 1 respectively).4244 Whereas the modal
lates were strongly related to clinical, environmental MICs mirror those found for C. neoformans, those for C.
and veterinary isolates from the west coast of the gattii s.l. did not. In particular for C. gattii s.l., adop-
USA, which suggests that these patients acquired the tion of the CLSI ECVs for our data set would bisect the
infection while travelling outside Denmark (Fig. 2). distributions and lead to a random number of isolates
Isolate DK0022 appeared to be C. deuterogattii (geno- with MICs above the ECV.
type AFLP6/VGII) and genetically closely related to In conclusion, the epidemiology of cryptococcal
clinical and environmental isolates from mainly South infections in Denmark reflects largely the Western
America (Fig. 2). As for the C. bacillisporus isolates, European situation with being C. neoformans genotype
this implies that the patient probably acquired the AFLP1/VNI as the major cause of disease, followed by
C. deuterogattii infection outside Denmark. This has C. deneoformans and C. gattii s.l. The latter were found
been reported before in a Danish traveller who shortly to be genetically closely related with those originating
visited Vancouver Island, British Columbia, Canada, from endemic regions, suggesting that the Danish
and developed a cryptococcal infection weeks after patients acquired their infection while travelling
returning to Denmark. The infecting isolate CBS10485 outside of Europe. Antifungal susceptibility testing
was genetically indistinguishable from the so-called showed species-specific differential susceptibility, but
Vancouver Island outbreak isolates (Fig. 2).27 Similar suggested that acquired resistance was an infrequent
imported cases of C. deuterogattii infections due to tra- phenomenon.
vel to the endemic area in Canada have been reported
before.3840 Acknowledgments
Antifungal susceptibility testing data for Cryptococ-
cus by the EUCAST methodology is scarce. Zaragoza J.F.M. received grants form Astellas, Merck and Basi-
and colleagues tested 10 C. neoformans s.l. and five lea. He has been a consultant to Basilea and Merck
C. gattii s.l. following EUCAST methodology and found and received speaker fees from Merck, Pfizer, United
amphotericin B MIC ranges of 0.030.125 mg l 1, Medical and Gilead. M.C.A. received speaker honoraria
flucytosine MICs 216 mg l 1, fluconazole MICs over the past 5 years from Astellas, Basilea, Gilead,
164 mg l 1 and voriconazole 0.0150.25 mg l 1 MSD, Pfizer and T2Biosystems and received research
and as presented here with higher fluconazole MICs grants/support (company/investigator initiated) paid to
for C. gattii s.l.41 Epidemiological cut-off values and the institution from Astellas, Basilea, Gilead and
clinical breakpoints have not yet been established by T2Biosystems.
neither EUCAST nor CLSI, and hence categorisation
into susceptible, intermediate and resistant is not pos- References
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8 2016 Blackwell Verlag GmbH


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