Medical Mycology 2001, 39, 185–193 Accepted 29 August 2000

PCR Ž ngerprinting: a convenient molecular tool to

distinguish between Candida dubliniensis and Candida
albicans
W. MEYER, K. MASZEWSKA & T. C. SORRELL
Centre for Infectious Diseases and Microbiology, Molecular Mycology Laboratory, The University of Sydney at Westmead Hospital,

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Sydney, Australia

Candida dubliniensis was recently identiŽed as a germ-tube- and chlamydospore-

positive yeast, phenotypically and morphologically indistinguishable from the
phylogenetically closely related yeast species C. albicans and its synonymized
variant C. stellatoidea. The high similarity between these yeast species causes
signiŽcant problems in the correct identiŽcation of C. dubliniensis in a standard
clinical mycology laboratory. Polymerase chain reaction (PCR) Žngerprinting
was successfully applied here to distinguish between clinical isolates of the
two closely related species. Microsatellite ([GACA]4 ) and minisatellite
([50 -GAGGGTGGCGGTTCT-30 ], derived from the core-sequence of the wild-type
phage M13) speciŽc oligonucleotides were used as single primers in PCR to amplify
hypervariable inter-repeat DNA sequences from 16 C. dubliniensis strains and 11
C. albicans strains. Each species, represented by its ex-type strain, could be identiŽed
by a distinct species-speciŽc multilocus pattern, allowing identiŽcation to species
level for all clinical isolates. In addition, the PCR Žngerprinting generated strain-
speciŽc proŽles, making this method applicable to epidemiological investigations.
PCR Žngerprinting using the primer M13 is proposed here as a simple, reliable and
highly reproducible molecular tool to differentiate between strains of C. albicans
and C. dubliniensis.
Keywords Candida albicans, Candida dubliniensis, identiŽcation, PCR Žnger-
printing

Introduction oral cavities [1–7], but isolation from other anatomical

sites (vagina and lung) has also been reported [1,8]. It
Since its Žrst description in 1995 [1], infection by
has been shown that C. dubliniensis can be a constituent
Candida dubliniensis has been increasingly reported in
of the normal human oral ora, with the potential
a number of human immunodeŽciency virus (HIV)-
to cause oral candidosis [1,8]. C. dubliniensis expresses
positive individuals and acquired immune deŽciency
the C. albicans serotype A [1,4] and is able to form
syndrome (AIDS) patients from around the world. In
germ tubes, pseudohyphae and abundant chlamydos-
these individuals it has been primarily isolated from the
pores [1,3,8]. These features have been diagnostic for
C. albicans, making it phenotypically and morpho-
logically indistinguishable from this phylogenetically
Correspondence: Dr Wieland Meyer, Centre for Infectious Dis- most closely related yeast species. In comparison to
eases and Microbiology, Molecular Mycology Laboratory, The C. albicans, C. dubliniensis isolates are characterized by
University of Sydney at Westmead Hospital, ICPMR, Level 3,
Room 3114A, Darcy Road, Westmead, NSW 2145, Australia.
a higher resistance to the commonly used antifungal drug
Tel.: ‡61 2 98456895; fax: ‡61 2 98915317; e-mail: meyer@ uconazole, and uconazole-susceptible isolates are able
angis.usyd.edu.au to develop resistance to the drug in vitro [1,9]. For this
ã ISHAM, Medical Mycology, 39, 185–193
2001 ISHAM
 186 Meyer et al.
reason it is important to identify clinical isolates
correctly at a very early stage of infection.
Despite the phenotypical similarities between these
two fungal pathogens, C. dubliniensis differs slightly in
its carbohydrate assimilation proŽle, as determined with
the commercially available biochemical yeast identiŽ ca-
tion systems (API ID 32C and API 20C AUX kits from
bioMérieux, Marcy l’Etoile, France) [10], in particular
the absence of ß-glucosidase [1,2]. However, these assays
are sometimes difŽcult to interpret. C. dubliniensis also
differs from C. albicans in its inhibited growth at
elevated temperatures of 42 oC [1,3,8] or 45 oC [11], its
growth on the recently developed commercial chromo-
genic agar medium CHROMagar Candida [12], where it
forms dark green colonies, and the absence of uores-
cence in the colonies on methyl blue-Sabouraud agar
plates [13]. However, individual strain variations have
been reported for each of these characteristics [1,3,8],
making these criteria unreliable for a correct identiŽ ca-
tion.
Because of the difŽculties in using methods based
on phenotypic characteristics to distinguish between
C. dubliniensis and C. albicans, the focus has shifted in
the past few years to molecular methods, which exploit
genetic differences between the two fungal pathogens to
develop more efŽcient differentiation techniques. These
techniques range from restriction fragment length
polymorphism (RFLP) generated using the enzymes
HaeIII and HinfI [1,14], to DNA-hybridization Žnger-
printing using the C. albicans-speciŽc repeat sequence
27A [1,15] and synthetic oligonucleotide probes homo-
logous to eukaryotic microsatellite sequences [1]. Other
techniques used are: random ampliŽed polymorphic
DNA (RAPD) analysis [1], sequence analysis of the
V3 region of the large ribosomal subunit gene
[1,16], electrophoretic karyotyping [1], and speciŽc
ampliŽcations of certain genes by polymerase chain
reaction (PCR) [17]. The most convincing evidence that
C. dubliniensis is a distinct taxon from C. albicans has
been the number of nucleotide differences (13–15
nucleotides) in the ribosomal RNA gene sequence,
when compared with C. albicans serotype A and B
strains as well as strains representing the synonym
C. stellatoidea [1,16,18] . However, most of these
techniques are labour intensive, cumbersome and
time-consuming. In spite of the identiŽ ed differences,
a rapid and accurate discrimination between C. albicans
and C. dubliniensis remains problematic in the average
clinical mycology laboratory.
In the original studies on C. dubliniensis by Sullivan et
al. [1], a number of microsatellite-speciŽ c oligonucleo-
tides (e.g. [GTG]5, [GACA]4 and [GATA]4) were used
as hybridization probes in DNA-Žngerprinting. Over the
past 6 years, our group has successfully shown that the
same oligonucleotides can be used as single primers in
PCR-Žngerprinting experiments to obtain highly infor-
mative multilocus proŽles from a number of fungal
genera [19–24]. In these studies a number of synthetic
oligonucleotides have been tested as single primers:
the core sequence of the wild-type phage M13
(50 -GAGGGTGGCGGTTCT-30 ), which is speciŽc to
minisatellite DNA sequences, and (CT)8, (GTG)5,
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(GACA)4 and (GATA)4, which are speciŽc to simple
repetitive DNA (microsatellite) sequences. It was found
that the primers M13 and (GACA)4 generate the most
discriminatory and informative Žngerprint proŽles. In
earlier studies, our group established the potential of this
PCR based technique to be used as a method for a fast
and reliable differentiation of several anamorphic
species of the yeast genus Candida and its associated
teleomorphic species. All investigated yeast species
could be clearly differentiated based on the unique
species-speciŽc banding pattern when these two primers
were used as single primers in the PCR [19,21]. In
addition the generated PCR Žngerprinting proŽles
displayed variation at the strain level, allowing the
separation of individual strains within each species
[19,21–23]. It was also demonstrated that the PCR
Žngerprints are stable in vitro and in vivo [22]. All of
the Žndings mentioned above make this technique an
ideal tool for a fast and reliable identiŽ cation of clinical
fungal specimens.
Based on the these Žndings, PCR Žngerprinting was
applied with the objective of differentiating the phylo-
genetically closely related yeast species C. albicans and
its variant C. stellatoidea from the recently described
species C. dubliniensis.
Materials and methods
Fungal isolates
Sixteen C. dubliniensis isolates, including the ex-type
culture (CBS 7987 = NCPF 3949), 10 C. albicans isolates,
including the ex-neotype culture, which is serotype A
(CBS 562) and a representative serotype B isolate (CBS
5983), as well as the ex-neotype culture for the
conspeciŽc variant C. stellatoidea (CBS 1905) (Table 1)
were examined in this study. The isolates were obtained
from the Centraalbureau voor Schimmelcultures (CBS,
Delft, The Netherlands), the Clinical Mycology Labora-
tory at Westmead Hospital, Westmead, Australia,
Sullivan & Nicolaides Pathology, Brisbane, Australia
and the Mycology Unit, Microbiology Department,
Auckland Hospital, Auckland, New Zealand. Cultures
were grown on yeast peptone glucose (YPD) agar plates
at 27 oC prior to DNA isolation. All isolates were
ã 2001 ISHAM, Medical Mycology, 39, 185–193
 PCR Žngerprinting of Candida dubliniensis 187

Table 1 List of type cultures and clinical isolates used in this study

Species/isolate Alternative number* Geographical origin Year of isolation Comments

Candida albicans (Robin) Berkhout (1923)

WM 83 Sydney/Australia 1995 HIV¡, from oral cavity
WM 204 Sydney/Australia Unknown
WM 205 WH 96-250-1065 Sydney/Australia 1996 From sputum
WM 211 WH 95-201-1014 Sydney/Australia 1995
WM 212 WH 96-256-0566 Sydney/Australia 1996 From necrotic tissue
WM 229 CBS 562 Uruguay 1935 NT, Serotype A, from skin

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WM 231 CBS 5983 Probably USA Unknown Serotype B
WM 622 ACC3243 Auckland/New Zealand 1998 HIV¡, from a mouth swab
WM 623 MA9026 Auckland/New Zealand 1992
WM 772 WH 99-105-2447 Sydney/Australia 1999 HIV¡, from sputum
Candida albicans synonym Candida stellatoidea (Jones & Martin) Langeron & Guerra (1939)
WM 230 CBS 1905 Unknown Unknown NT
Candida dubliniensis Sullivan (1995)
WM 206 WH 96-241-1055 Sydney/Australia 1996 From sputum
WM 602 CBS 7987 Dublin/Ireland 1995
NCPF 3949
WM 606 CBS 7988 Melbourne/Australia 1995 HIV¡, from oral cavity
WM 607 CBS 8500 Nijmegen, Netherlands 1998 From blood
WM 608 CBS 8501 Nijmegen, Netherlands 1998
WM 609 WH 98-079-1183 New York, USA 1998 HIV‡, from oral cavity
WM 611 WH 96-212-2001 Sydney/Australia 1998 From throat
WM 615 MD8127 Auckland/New Zealand 1998 HIV¡, from sputum
WM 616 SH3240 Auckland/New Zealand 1998 HIV¡, from sputum
WM 617 BS5206 Auckland/New Zealand 1997 HIV¡, from sputum
WM 618 BY9374 Auckland/New Zealand 1998 HIV¡, from sputum
WM 619 BI3140 Auckland/New Zealand 1996 HIV¡, from abdominal uid
WM 620 SG8798 Auckland/New Zealand 1997 HIV¡, from throat swab
WM 621 CB1830 Auckland/New Zealand 1998 HIV¡, from lip swab
WM 624 WH 99-025-1889 Sydney/Australia 1999 From sputum
WM 771 BC901 Brisbane/ Australia 1999 HIV‡, from blood

CBS, Centraalbureau voor Schimmelcultures, Baarn-Delft, The Netherlands; WH, Westmead Hospital, The University of Sydney, Sydney, Australia; T, ex-type
culture; NT, ex-neotype culture.
*, Numbers deŽning the same strain in a different culture collection are given under alternative number column.

maintained on Sabouraud agar slants at 4 oC, as water
cultures at room temperature and as glycerol stocks at
¡70 oC.
PCR Žngerprinting
Genomic DNA was isolated as described previously [21].
PCR Žngerprinting was carried out using oligonucleo-
tides of the minisatellite speciŽc core sequence of the
wild-type phage M13 (50 -GAGGGTGGCGGTTCT-30 )
[25] and of the microsatellite (simple repetitive DNA
sequence) (GACA)4 [26] as single primers. AmpliŽca-
tion reactions were performed in 50 m l volumes contain-
ing 25 ng of genomic DNA template, 10 mM Tris-HCl,
pH 8¢3, 50 mM KCl, 1¢5 mM MgCl, 0¢2 mM each of dATP,
dCTP, dGTP and dTTP (Boehringer Mannheim, Man-
nheim, Germany), 3 mM magnesium acetate, 30 ng
primer and 2¢5 units of AmpliTaq DNA polymerase
(Perkin Elmer, Norwalk, CT, USA). The PCR reactions
were performed in a Perkin Elmer thermal cycler (model
ã 2001 ISHAM, Medical Mycology, 39, 185–193
480) with 35 cycles of 20 s denaturation at 94 oC, 1 min
annealing at 50 oC for the primers M13 and (GTG)5, and
at 43 oC for the primer (GACA)4, and 20 s extension at
72 oC, followed by a Žnal extension cycle for 6 min at
72 oC. The PCR products were removed, concentrated
to approximately 20 m l (Eppendorf Concentrator, model
5301; Eppendorf-Netheler GmbH, Hamburg, Germany),
and separated by electrophoresis in 1¢4% agarose gels in
Tris-borate-EDTA buffer for 10 h at 3 V cm¡1. Ampli-
Žcation products were detected by staining with ethi-
dium bromide (0¢5 m g ml¡1) and visualized under UV
light. The 1 kb DNA ladder from GIBCO-BRL was used
as molecular size standard.
Analysis of genetic relatedness
PCR Žngerprinting proŽles were analysed using the
GelComparII version 1.01 software (Applied Maths,
Kortrijk, Belgium) [27]. All visible bands were included
 188 Meyer et al.

in the analysis regardless of the band intensity. The
bands for each Žngerprinting pattern were deŽned using
the manual option in the GelComparII software with a
band tolerance of 1%. This was the minimum position
tolerance that was required to normalize the molecular
size markers to 100% identity. Similarity coefŽcients
were calculated using the dice algorithm, and cluster
analysis performed by the unweighted pair group
method for arithmetic averages (UPGMA). For epide-
for C. dubliniensis (Fig. 1a). The bands obtained
with the primer (GACA)4 ranged 600–2040 bp for
C. albicans and 400–4000 bp for C. dubliniensis (Fig. 2a).
All clinical isolates could be clearly assigned to one or
the other species based on the species-speciŽc banding
patterns obtained.
C. albicans serotypes A and B, and the C. stellatoidea
showed similar PCR Žngerprinting proŽles, with a band
sharing similarity of 70% for both primers used (Figs. 1a

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miological purposes the Žngerprinting data obtained
using the primers M13 or (GACA)4 were combined as a
composite data set and corrected for internal weights, to
produce cluster analysis based upon the combined
Žngerprint data.
Results and discussion
The current study includes the ex-type or ex-neotype
cultures of C. albicans, C. stellatoidea and C. dubliniensis,
as well as several clinical isolates. All the clinical isolates
studied were tentatively identiŽed as C. dubliniensis, on
the basis of giving an atypical C. albicans identiŽcation
proŽle using commercially available biochemical identi-
Žcation systems (API ID32C; bioMérieux) and failing to
grow at elevated temperatures but producing germ tubes
and chlamydospores.
PCR Žngerprinting was carried out using the primers
M13 or (GACA)4 as single primers to amplify
hypervariable inter-repeat DNA fragments from all
investigated yeast strains. The PCR proŽles obtained
were highly informative and generated clearly distinct
banding patterns for the C. albicans and C. dubliniensis
isolates (Figs. 1a and 2a). The same PCR based
techniques were used previously in our laboratory to
distinguish other medically important yeasts [19,21–23].
The primer M13 generated DNA fragments ranging in
sizes of 500–1900 bp for C. albicans and 350–2500 bp
and 2a). These band sharing values are within the intra-
species range of variation (70–85%) observed previously
for other Candida species [19,21], conŽrming that the
species are conspeciŽc in spite of differing in one or two
nucleotides in the V3 region of the ribosomal RNA gene
[18,28,29]. The differences observed between the Žnger-
printing patterns (Figs. 1a and 2a) are due to strain-
speciŽc variation within a single species [19,21].
Major discriminatory or diagnostic Žngerprinting
bands for each species were identiŽed and their
molecular weights determined using the computer
program GelComparII version 1.01, based upon Žnger-
print patterns representing each of the isolates in this
study. Most of these major bands were present in all
isolates studied (Figs. 1a and 2a). The major discrimina-
tory bands are presented in Table 2 and arrow heads in
Figures 1a and 2a indicate their locations within the
multilocus proŽles, obtained with the two primers
respectively.
Similarity coefŽcients for all possible pairs of isolates
were calculated using the Dice algorithm with a band
position tolerance of 1% and dendrograms were
generated by the UPGMA analysis. These dendrograms
split the investigated yeast isolates into two groups.
Isolates representing C. albicans and its conspeciŽc
variant formerly known as C. stellatoidea form one
cluster clearly separated from the isolates clustered with
the ex-type culture of C. dubliniensis, which form a
second distinct cluster. All of the clinical isolates are

Table 2 Molecular sizes of major discriminatory/diagnostic PCR Žngerprinting bands of C. albicans and C. dubliniensis, for the primers M13
and (GACA)4

Primer Species No. of major Molecular sizes of major bands (bp)*

diagnostic bands

M13
C. albicans 9 1800y, 1670, 1420, 1170, 1120, 1050y, 750, 710, 605
C. dubliniensis 11 2330, 1960, 1825, 1410, 1120, 1090, 907y, 765, 654y, 535, 375
(GACA)4
C. albicans 7 1975, 1890, 1255, 1160, 1065, 865, 835
C. dubliniensis 13 4150, 3620, 3270, 2290, 1600y, 1450y, 1400y, 1150, 1010, 860, 815, 730, 540y

*, Molecular sizes of the PCR Žngerprint bands were determined automatically from computer-scanned photographs of agarose gels by comparison with the
molecular size standard (1 kb marker) using the computer program GelComparII version 1.01;
y, major diagnostic PCR-Žngerprinting bands which may not be present in all investigated strains.

ã 2001 ISHAM, Medical Mycology, 39, 185–193

 PCR Žngerprinting of Candida dubliniensis 189

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Fig. 1 (a) Electrophoretic separation of PCR Žngerprints obtained by amplifying genomic DNA from a number of type, neotype and
clinical isolates of C. albicans and C. dubliniensis using the M13 core sequence (50 -GAGGGTGGCCGGTTCT-30 ) as a single primer in the
PCR. For full description of the investigated isolates, see Table 1. Major discriminatory/diagnostic PCR Žngerprinting bands are indicated by
arrows and values given in Table 2. (b) Dendrogram derived from the similarity matrix obtained from the PCR-Žngerprint pattern generated
with the primer M13 after analysis with the program GelComparII version 1.01 using Dice coefŽcient with a band tolerance of 1% and
UPGMA analysis.
ã 2001 ISHAM, Medical Mycology, 39, 185–193
 190 Meyer et al.

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Fig. 2 (a) Electrophoretic separation of PCR Žngerprints obtained by amplifying genomic DNA from a number of type, neotype and
clinical isolates of C. albicans and C. dubliniensis using (GACA)4 as a single primer in the PCR. For full description of the investigated
isolates, see Table 1. Major discriminatory/diagnostic PCR Žngerprinting bands are indicated by arrows and values given in Table 2.
(b) Dendrogram derived from the similarity matrix obtained from the PCR Žngerprint pattern generated with the primer (GACA)4 after
analysis with the program GelComparII version 1.01 using Dice coefŽcient with a band tolerance of 1% and UPGMA analysis.

ã 2001 ISHAM, Medical Mycology, 39, 185–193


grouped correctly with their respective standard ex-type
or ex-neotype strains (Figs. 1b and 2b). The computer
scanned PCR proŽles generated in this study and from
other Candida species in previous studies [19,21] have
formed the basis of a computer database, which can be
used for future identiŽ cations of atypical or unidentiŽ-
able Candida isolates.
The band tolerance of 1% used in the present analysis
was determined during computer analysis of the mole-
cular weight standard (1 kb ladder; GIBCO-BRL),
which was included at three intervals on each gel. It
represents the tolerance required for corresponding
bands in each molecular weight standard to be inter-
preted as identical in the dendrogram, compensating for
small, unavoidable differences in the running conditions
of each gel. No further computer-enhanced optimiza-
tions were made.
The results presented in this study demonstrate again
that PCR Žngerprinting data do not reect deŽnitive
phylogenetic relationships between a given set of isolates
[21]. This technique provides an indication of the
relationships of isolates according to the primer/PCR
system utilized (Figs. 1b and 2b). To overcome the fact
that each primer ampliŽes only a small independent
Fig. 3 Dendrogram generated form the
composite data set after combining the
PCR Žngerprinting data obtained with the
primers M13 and (GACA)4 , using internal
weights deŽned in the individual GelCom-
parII analysis for each primer, showing the
genetic relationships between the indivi-
dual isolates.
ã 2001 ISHAM, Medical Mycology, 39, 185–193
PCR Žngerprinting of Candida dubliniensis 191
subset of the genome which is not representative of the
true phylogenic relationships between the investigated
isolates, the obtained data sets have to be combined for
epidemiological purposes to obtain accurate genetic
relationships (Fig. 3).
In addition to correctly identifying each of the clinical
isolates to the species level, the primers used were also
able to distinguish all investigated isolates. The average
intra-species variation of the PCR Žngerprints obtained
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with the primer M13 was 18¢3% (standard deviation of
7¢7) for C. albicans/C. stellatoidea and 10¢4% (standard
deviation of 6¢8) for C. dubliniensis. With primer
(GACA)4, there was an average intra-species variation
of 18¢3% (standard deviation of 7¢4) for C. albicans/
C. stellatoidea and 15¢9% (standard deviation of 12¢7) for
C. dubliniensis.
The current analysis revealed that the minisatellite
speciŽc primer M13 is more discriminatory than the
simple repetitive DNA sequence (microsatellite) speciŽc
primer (GACA)4. The primer M13 could distinguish all
isolates investigated. With primer (GACA)4, three
isolates appeared to be identical even though there was
no apparent correlation between the patients they came
from. One such isolate was from the USA (WM609)
 192 Meyer et al.
while two were from New Zealand (WM618 and
WM619), isolated from different patients several years
apart. These Žndings again emphasize that it is important
to use at least two independent markers for the
identiŽcation of any given clinical isolate, in order to
overcome the bias caused by differences in distribution
and rates of evolution of certain loci.
The fact that the presented identiŽcation system
allows both species and strain identiŽ cation differenti-
ates it from a number of previously suggested identiŽ ca-
tion systems that only identify isolates to the species
level [17,29]. The main difference of the proposed PCR
Žngerprinting technique is that the primers used are
speciŽc to mini- and microsatellites, genetic elements
that are found in a wide range of eukaryotic organisms.
That is why the same primers and PCR conditions can be
used for the identiŽ cation of a wide range of fungal
pathogens. The species-speciŽc major banding patterns
generate a unique species identiŽ cation pattern. The
identiŽcation power is only limited by the size of the
available databases (meaning the number of included
species). Previously described molecular methods were
only applicable to single species or to a small range of
species they had been speciŽcally developed for. In
addition to circumventing this limitation, the PCR
Žngerprinting technique is also a very useful method
for epidemiological investigations, particularly for the
identiŽcation of nosocomial outbreaks and of drug
resistant strains, if the independent Žngerprinting data
sets are analysed in a combined fashion (Fig. 3). The
proposed identiŽcation technique is simple, reliable and
does not require radioactively labelled probes, such as
those recently developed for C. dubliniensis [30], making
it an ideal identiŽ cation system for routine clinical
mycology laboratories.
The close phylogenetic relationship between the two
yeast species is demonstrated in the relatively high inter-
species homology of their Žngerprinting proŽles. The
observed inter-species homology was 57¢9% for the
primer M13 and 58¢6% for the primer (GACA)4,
compared with previously obtained data from other
yeast species, which were characterized by 15–30% inter-
species homology [14,21,22].
We propose PCR-Žngerprinting using especially the
primer M13 as a simple, rapid, stable, sensitive, highly
reproducible and cost effective molecular tool to
distinguish C. albicans and C. dubliniensis.
Acknowledgements
We thank Ok Cha Lee, Clinical Mycology Laboratory,
Westmead Hospital, Westmead, Australia, for the
biochemical yeast identiŽ cation, Heide-Marie Daniel
for maintenance of cultures and preparation of photo-
graphs and Sarah Kidd, both Molecular Mycology
Laboratory, Westmead Hospital, for proofreading the
manuscript. We appreciate the gifts of cultures from:
David Yarrow, CBS; Ok Cha Lee, Jenny Robson and
Susan Benson, Sullivan & Nicolaides Pathology, Bris-
bane, Australia, and Dinah Parr and Karen Rogers,
Mycology Unit, Microbiology Department, Auckland
Hospital, Auckland, New Zealand. This investigation
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was supported by an NH&MRC CARG grant (#960792)
from the National Health & Medical Research Council,
Canberra, Australia to W. Meyer, and two grants-in-aid
from the Millennium Foundation of the Westmead
Institute of Health Research, Westmead, Australia to
W. Meyer.
References
1 Sullivan DJ, Westerneng TJ, Haynes KA, Bennett DE, Cole-
man DC. Candida dubliniensis sp. nov.: phenotypic and
molecular characterization of a novel species associated with
oral candidosis in HIV-infected individuals. Microbiol 1995; 141:
1507–1521.
2 Boerlin P, Boerlin-Pietzold F, Durussel C, et al. Cluster of oral
atypical Candida isolates in a group of human immunodeŽ-
ciency virus-positive drug users. J Clin Microbiol 1995; 33: 1129–
1135.
3 Coleman DC, Sullivan DJ, Bennett DE, et al. Candidiasis: the
emergence of a novel species Candida dubliniensis. AIDS 1997;
11: 557–567.
4 Coleman D, Sullivan D, Haynes K, et al. Molecular and
phenotypic analysis of Candida dubliniensis: a recently identi-
Žed species linked with oral candidoses in HIV-infected and
AIDS patients. Oral Dis 1997; 3 (Suppl. 1): S96–S101.
5 Jabra-Ritz MA, Baqui AAMA, Kelley JI, et al. IdentiŽcation of
Candida dubliniensis in a prospective study of patients in the
United States. J Clin Microbiol 1999; 37: 321–326.
6 McCullough M, Ross B, Reade P. Characterization of geneti-
cally distinct subgroup of Candida albicans strains isolated from
oral cavities of patients infected with human immunodeŽciency
virus. J Clin Microbiol 1995; 33: 696–700.
7 Sullivan D, Haynes K, Bille J, et al. Widespread geographic
distribution of oral Candida dubliniensis strains in human
immunodeŽciency virus-infected individuals. J Clin Microbiol
1997; 35: 960–964.
8 Sullivan D, Coleman D. Candida dubliniensis: characteristics
and identiŽcation. J Clin Microbiol 1998; 36: 329–334.
9 Moran GP, Sullivan DJ, Henman MC, et al. Antifungal drug
susceptibilities of oral Candida dubliniensis isolates from human
immunodeŽciency virus (HIV)-infected and non-HIV-infected
subjects and generation of stable uconazole-resistant deriva-
tives in vitro. Antimicrob Agents Chemother 1997; 41: 617–623.
10 Salkin IF, Pruitt WR, Padhye AA, et al. Distinctive carbohy-
drate assimilation proŽles used to identify the Žrst clinical
isolates of Candida dubliniensis recovered in the United States.
J Clin Microbiol 1998; 36: 1467.
11 Pinjon E, Sullivan D, Salkin I, Shanley D, Coleman D. Simple,
inexpensive, reliable method for differentiation of Candida
dubliniensis from C. albicans. J Clin Microbiol 1998; 36: 2093–
2095.
ã 2001 ISHAM, Medical Mycology, 39, 185–193

12 Odds FC, Bernaerts R. CHROMagar Candida, a new differ-
ential isolation medium for presumptive identiŽcation of
clinically important Candida species. J Clin Microbiol 1994;
32: 1920–1929.
13 Schoofs A, Odds FC, Colebunders R, Leven L, Gossens H. Use
of specialised isolation media for recognition and identiŽcation
of Candida dubliniensis isolates from HIV-patients. Eur J Clin
Microbiol Infect Dis 1997; 16: 296–300.
14 McCullough M, Clemons KV, Stevens DA. Molecular and
phenotypic characterization of genotypic Candida albicans
subgroups and comparison with Candida dubliniensis and
Candida stellatoidea. J Clin Microbiol 1999; 37: 417–421.
15 Scherer S, Stevens DA. A Candida albicans dispersed, repeated
gene family and its epidemiologic applications. Proc Natl Acad
Sci USA 1988; 85: 1452–1456.
16 GilŽllan GD, Sullivan DJ, Haynes K, et al. Candida dubliniensis:
phylogeny and putative virulence factors. Microbiol 1998; 144:
829–838.
17 Kurzai O, Heinz WJ, Sullivan DJ, et al. Rapid PCR test for
discriminating between Candida albicans and Candida dubli-
niensis isolates using primers derived from the pH-regulated
PHR1 and PRH2 genes of C. albicans. J Clin Microbiol 1999, 37:
1587–1590.
18 Meyer SA. DNA relatedness between physiologically similar
strains and species of yeasts of medical and industrial
importance. In: Garrattini S, Paglialunga S, Scrimshaw NS,
eds. Investigation of Single Cell Protein. New York: Pergamon
Press, 1979: 13–19.
19 Latouche GN, Daniel HM, Lee OC, et al. Comparison of use of
phenotypic and genotypic characteristics for identiŽcation of
species of the anamorph genus Candida and related teleomorph
yeast species. J Clin Microbiol 1997; 35: 3171–3180.
ã 2001 ISHAM, Medical Mycology, 39, 185–193
PCR Žngerprinting of Candida dubliniensis 193
20 Lieckfeldt E, Meyer W, Börner T. Rapid identiŽcation and
differentiation of yeasts by DNA and PCR-Žngerprinting. J
Basic Microbiol 1993; 33: 413–426.
21 Meyer W, Latouche GN, Daniel HM, et al. IdentiŽcation of
pathogenic yeasts of the imperfect genus Candida by PCR-
Žngerprinting. Electrophoresis 1997; 18: 1548–1559.
22 Meyer W, Mitchell TG. Polymerase chain reaction Žngerprint-
ing in fungi using single primers speciŽc to minisatellites and
simple repetitive DNA sequences: strain variation in Crypto-
coccus neoformans. Electrophoresis 1995; 16: 1648–1656.
23 Meyer W, Mitchell TG, Freedman EZ, Vilgalys R. Hybridiza-
Downloaded from https://academic.oup.com/mmy/article-abstract/39/2/185/986647 by guest on 30 March 2019
tion probes for conventional DNA Žngerprinting used as single
primers in the polymerase chain reaction to distinguish strains of
Cryptococcus neoformans. J Clin Microbiol 1993; 31: 2274–2280.
24 Weising K, Nybom H, Wolff K, Meyer W. DNA Fingerprinting
in Plants and Fungi. Boca Raton: CRC Press, 1995.
25 Vassart G, Georges M, Monsieur R, et al. A sequence in M13
phage detects hypervariable minisatellites in human and animal
DNA. Science 1987; 235: 683–684.
26 Ali S, Müller CR, Epplen JT. DNA Žngerprinting by
oligonucleotide probes speciŽc to simple repeats. Hum Genet
1986; 74: 239–243.
27 Applied Maths BVBA. GelCompar II version 1.01. Kortrijk,
Belgium, 1999.
28 Kwon-Chung KJ, Riggsby WS, Uphoff RA, et al. Genetic
differences between type I and type II Candida stellatoidea.
Infect Immun 1989; 57: 627–532.
29 Montrocher R, Claisse ML. Biochemical studies in the yeast
genus Candida. Cell Mol Biol 1980; 30: 291–301.
30 Joly S, Pujol C, Rysz M, Vargas K, Soll DS. Development and
characterization of complex DNA Žngerprinting probes for the
infectious yeast Candida dubliniensis. J Clin Microbiol 1999; 37:
1035–1044.