Performance comparison of phenotypic and molecular methods for detection and differentiation of Candida albicans and Candida dubliniensis
BMC Infectious Diseases
Performance comparison of phenotypic and molecular methods for detection and differentiation of Candida albicans and Candida dubliniensis
Suhail Ahmad 0
Ziauddin Khan 0
Mohammad Asadzadeh 0
Ajmal Theyyathel 0
Rachel Chandy 0
0 Department of Microbiology, Faculty of Medicine, Kuwait University , P. O. Box 24923, Safat 13110 , Kuwait
Background: Candida albicans is the most pathogenic Candida species but shares many phenotypic features with Candida dubliniensis and may, therefore, be misidentified in clinical microbiology laboratories. Candidemia cases due to C. dubliniensis are increasingly being reported in recent years. Accurate identification is warranted since mortality rates are highest for C. albicans infections, however, C. dubliniensis has the propensity to develop resistance against azoles more easily. We developed a duplex PCR assay for rapid detection and differentiation of C. albicans from C. dubliniensis for resource-poor settings equipped with basic PCR technology and compared its performance with three phenotypic methods. Methods: Duplex PCR was performed on 122 germ tube positive and 12 germ tube negative isolates of Candida species previously identified by assimilation profiles on Vitek 2 ID-YST system. Typical morphologic characteristics on simplified sunflower seed agar (SSA), and reaction with a commercial (Bichro-Dubli) latex agglutination test were also performed. The assay was further applied on 239 clinical yeast and yeast-like fungi and results were confirmed by DNA sequencing of internal transcribed spacer (ITS) region of rDNA. Results: The results of duplex PCR assay for 122 germ tube positive and 12 germ tube negative isolates of Candida species were comparable to their identification by Vitek 2 ID-YST system, colony characteristics on SSA and latex agglutination test. Application of duplex PCR also correctly identified all 148 C. albicans and 50 C. dubliniensis strains among 239 yeast-like fungi. Conclusions: The data show that both, duplex PCR and Bichro-Dubli are reliable tests for rapid (within few hours) identification of clinical yeast isolates as C. dubliniensis or C. albicans. However, duplex PCR may be applied directly on clinical yeast isolates for their identification as C. dubliniensis or C. albicans as it does not require prior testing for germ tube formation or latex Candida agglutination.
Candida albicans; Candida dubliniensis; Detection; Differentiation; Duplex PCR
Candida albicans is the predominant pathogenic Candida
species in susceptible human host. Infections caused by
non-albicans Candida spp. have, however, also increased
in recent years and may account for up to 60% of all
episodes of candidemia or invasive candidiasis at some centers
[1,2]. Candida dubliniensis, first described in 1995 from
oral cavities of human immunodeficiency virus-infected
individuals , is increasingly being reported from patients
with candidemia in recent years [1,2,4]. C. dubliniensis
shares several phenotypic characteristics, such as ability
to form chlamydospores on cornmeal agar and germ
tubes in serum with C. albicans. Since germ tube test is
routinely used for the differentiation of C. albicans from
other Candida species, identification based solely on this
test leads to misidentification of some C. albicans
isolates in routine diagnostic laboratories . Accurate
identification is warranted since C. dubliniensis exhibits
increased adherence to buccal epithelial cells and is more
likely to develop resistance against fluconazole and other
Several phenotypic tests based on colony morphology
and physiological assimilation tests have been developed
to distinguish C. albicans from C. dubliniensis.
Commercially available yeast identification systems (Vitek 2
IDYST, API 20C and ID32C) based on utilization of various
compounds have been used for differentiating various
Candida spp., however, these methods are expensive and
require at least 12 days to report results . Formation
of rough/fringed colonies by germ tube positive isolates
on differential media such as Niger seed agar ,
simplified sunflower seed agar (SSA) , or tobacco agar ,
or inability to grow in a hypertonic Sabouraud dextrose
broth containing 6.5 M NaCl  are often used to
differentiate C. albicans from C. dubliniensis. A commercial
latex agglutination test (Bichro-Dubli Fumouze) is also
available . However, prior presumptive identification
of C. albicans/C. dubliniensis is required before
phenotypic methods could be used for their differentiation. Also,
variations in growth conditions (incubation temperature,
repeated subculturing and storage) may impede accurate
identification of these two species by phenotypic tests.
Unambiguous identification can be established only by
molecular techniques and PCR-based methods are mostly
employed . Matrix-assisted laser desorption
ionizationtime of flight mass spectrometry (MALDI-TOF MS) has
also been exploited recently for identification and
differentiation of cultured yeast isolates including even closely
related Candida spp. . However, the high cost of the
equipment is a major impediment for its widespread use
in resource-limited settings. In this study, we have
developed a novel duplex PCR assay by using primers derived
from unique rDNA sequences for rapid detection and
differentiation of C. albicans and C. dubliniensis. The
performance of duplex PCR was compared with three
phenotypic methods for identification of clinical isolates
as C. albicans or C. dubliniensis.
Reference strains and clinical isolates
Candida dubliniensis (CD 36/CBS 7987/ATCC
MYA646), C. albicans (ATCC 90028), Candida parapsilosis
(ATCC 10233), Candida glabrata (ATCC 15545),
Candida tropicalis (ATCC 750) and Candida krusei (CBS
6258) were used as reference Candida species. One
hundred thirty-four clinical isolates representing C.
dubliniensis (n = 67), C. albicans (n = 55), C. parapsilosis
(n = 2), C. glabrata (n = 2), C. tropicalis (n = 2), Candida
lusitaniae (n = 2), Candida krusei (n = 2) and Candida
kefyr (n = 2) were tested by both phenotypic and
molecular tests. All isolates of C. albicans and C. dubliniensis
were positive by germ tube test. Identity of C.
dubliniensis strains was also confirmed by sequencing of internal
transcribed spacer (ITS) region (containing ITS-1, 5.8S
rRNA and ITS-2) of rDNA as described previously .
Additionally, 239 clinical yeast isolates including C.
albicans (n = 148), C. dubliniensis (n = 50), C. parapsilosis
(n = 5), C. glabrata (n = 5), C. lusitaniae (n = 5), C.
tropicalis (n = 5), Candida haemulonii (n = 3), Candida rugosa
(n = 2), Trichosporon species (n = 4) and Cryptococcus
species (n = 6) and one isolate each of C. krusei, Candida
guilliermondii, C. orthopsilosis, C. metapsilosis, C.
nivariensis and C. bracarensis were also tested by duplex PCR.
The isolates were cultured as part of standard patient
care and no individual patient approval was needed
for their subsequent use. The project under which the
study was performed was approved by the Ethical
Committee of Faculty of Medicine, Health Sciences center,
Phenotypic identification and characterization
Clinical isolates were speciated by carbohydrate
assimilation profile using Vitek 2 ID-YST system (bioMerieux,
Marcy-lEtoile, France) and tested for germ tube formation
in horse serum. All germ tube positive isolates were
presumptively identified as C. albicans/C. dubliniensis.
Germ tube positive isolates showing fringed/rough colonies
on SSA  were identified as C. dubliniensis.
BichroDubli (Fumouze Diagnotics, Levallois-Perret, France) latex
agglutination test for identification of C. dubliniensis
strains was performed on all germ tube positive isolates
according to manufacturers recommendations.
Template DNA preparation, duplex PCR assay and
For molecular studies, a loop full of yeast colony from
Sabouraud dextrose agar plate was suspended in 1 ml of
sterile water in a microcentrifuge tube containing 50 mg
Chelex-100 (Sigma-Aldrich Co.), the contents were
heated at 95C for 20 min and then centrifuged. The
supernatant was transferred to a new tube and typically
2 l was used in PCR. Species-specific identity of
C. albicans and C. dubliniensis strains was performed by
duplex PCR by using primers targeting sequences in
ITS-1 and ITS-2 regions of rDNA. Species-specificity
of primer pairs CALF
(50-TGGTAAGGCGGGATCGCTT-30) + CALR (50-GGTCAAAGTTTGAAGATATAC)
and CDUF (50-AAACTTGTCACGAGATTATTTTT) +
CDUR (50-AAAGTTTGAAGAATAAAATGGC-30) for
C. albicans and C. dubliniensis, respectively, was
indicated by BLAST searches (http://www.ncbi.nlm.nih.gov/
BLAST/Blast.cgi?). PCR amplification was performed in
a final volume of 50 l containing 1x AmpliTaq DNA
polymerase buffer I and 2 units of AmpliTaq DNA
polymerase (Perkin Elmer), 10 pmol of CALF + CALR +
CDUF + CDUR primers, 2 l of template DNA and
100 M of each dNTP. Cycling conditions included an
initial denaturation at 95C for 5 min followed by
30 cycles of 95C for 1 min, 55C for 30 s and 72C for
1 min and a final extension at 72C for 10 min. PCR
products (20 l) were run on 2% (w/v) agarose gels, as
described previously . The species-specific identity
of clinical yeast isolates was established by sequencing of
ITS region of rDNA. The ITS region was amplified by
using panfungal primers ITS1  and CTSR  and
the amplicons were purified and sequenced as described
in detail previously [15,19].
Duplex PCR amplification with CALF + CALR + CDUF +
CDUR primers yielded an amplicon of ~100 bp and
~325 bp only with DNA from reference strains of C.
albicans and C. dubliniensis, respectively, but not from
other Candida species, as expected (data not shown). Of
134 clinical Candida spp. isolates, 122 were positive for
germ tube formation and 12 were germ tube negative.
Vitek 2 ID-YST system identified the isolates as C.
dubliniensis (n = 67), C. albicans (n = 55), C. parapsilosis
(n = 2), C. glabrata (n = 2), C. tropicalis (n = 2), C.
lusitaniae (n = 2), C. krusei (n = 2) and C. kefyr (n = 2). Duplex
PCR correctly identified all 67 C. dubliniensis and 55 C.
albicans isolates (data from 11 selected isolates are
shown in Figure 1) while negative results were obtained
for the remaining 12 germ tube negative isolates
(Table 1). Growth on SSA correctly identified 67 of 122
germ tube positive isolates as C. dubliniensis.
BichroDubli test was positive for 64 of 67, weakly positive for 2
of 67 and negative for 1 of 67 isolates identified as C.
dubliniensis by Vitek 2 ID-YST system and ITS region
sequencing. Bichro-Dubli test was negative for 55 of 122
germ tube positive isolates identified as C. albicans by
Vitek 2 ID-YST system as well as 12 germ tube negative
Figure 1 Agarose gel of duplex PCR assay from representative
isolates of C. albicans (lanes 13, 5, 10, 11) and C. dubliniensis
(lanes 4, 69). No amplification was obtained in control (no DNA
added) tubes. The duplex PCR test was uniformly negative for other
Candida species or yeast-like fungi. Lane M is 100 bp DNA ladder
and the positions of migration of 100 bp, 300 bp and 600 bp
fragments are marked.
isolates of six other Candida species (Table 1). Thus,
duplex PCR accurately identified all 55 C. albicans and
67 C. dubliniensis strains among a panel of 134
wellcharacterized Candida spp. isolates with 100% sensitivity
Direct application of duplex PCR on a panel of 239
clinical isolates of yeast-like fungi correctly identified
all 148 C. albicans and 50 C. dublininsis strains. The
remaining isolates representing other Candida spp. or
other yeast-like fungi yielded negative results. The
results were confirmed by direct DNA sequencing of
ITS region of rDNA.
The results of duplex PCR assay described here were
completely concordant with species-specific
identification of C. dubliniensis and C. albicans strains by Vitek 2
ID-YST system and colony characteristics (fringed/rough
colonies) on SSA. However, both, Vitek 2 ID-YST system
and growth on SSA usually require 2448 hours before
results are available. Furthermore, identification of C.
dubliniensis strains on SSA requires prior testing for
germ tube formation while Vitek 2 ID-YST system is
expensive and thus, is not readily available in
resourcepoor settings . The requirement for minimal amount
of genomic DNA for duplex PCR allows the whole
procedure to be completed within 4 hours. Furthermore,
since duplex PCR employs species-specific primers for
both, C. albicans and C. dubliniensis, it can be directly
applied on clinical isolates of yeast and yeast-like fungi.
Indeed, direct application of duplex PCR on a panel of
239 clinical isolates of yeast-like fungi correctly
identified all 50 C. dublininsis and 148 C. albicans strains.
Table 1 Performance comparison of duplex PCR with Vitek 2 ID-YST system, Bichro-Dubli Fumouze latex agglutination
test and colony characteristics on simplified sunflower seed agar (SSA) for identification and differentiation of C.
albicans and C. dubliniensis
No. of isolates yielding correct identification with
*Fringed/rough colonies during growth on simplified sunflower seed agar (SSA).
Although several PCR-based methods using rDNA as
target have been described previously, they either involve
two separate PCR reactions for each strain [16,20,21] or
further manipulations (such as restriction digestion to
generate restriction fragment length polymorphism or
DNA sequencing) are needed for species-specific
identification [16,22-24]. These additional steps add to the
cost of the test and/or consume additional time thus
delaying results. Other investigators have used
PCRbased methods targeting intronic sequences for
differentiation of C. albicans from C. dubliniensis [25-27].
However, these approaches may lead to misidentification
of some isolates since intronic sequences may vary
significantly . A real-time PCR assay using melting
point analysis was described recently for detection and
differentiation of C. albicans and C. dubliniensis strains
but involves separate reactions for each Candida spp.
and expensive probe primers . Although two
realtime PCR assays using SYBR Green dye have also been
described, these assays either require prior presumptive
identification of C. albicans/C. dubliniensis strains by
another (such as germ tube) test and/or two rounds of
real-time PCR assays are needed [29,30]. Furthermore,
the difference in Tm values for C. albicans and C.
dubliniensis strains in the two reports was either small (<0.5C)
or negligible which may lead to misidentification of
Performance of another rapid (Bichro-Dubli Fumouze
latex agglutination) test was also compared with duplex
PCR results. The 98.5% sensitivity and specificity of
100% of Bichro-Dubli test is in agreement with the
reported sensitivity (97% to 100%) and specificity (100%)
values suggested by the kit manufacturer. Similar values
have also been reported in other studies for
identification of C. dubliniensis isolates [31,32]. Although
BichroDubli test is also a rapid method for identification of
C. dubliniensis, the test can only be applied on isolates
previously identified as C. albicans/C. dubliniensis either
by germ tube formation of by application of another
(Bichro Latex Albicans) latex agglutination test [13,32].
This increases both, the cost and the time to report
results for Bichro-Dubli test. The use of chromogenic
differential media such as CHROMagarW Candida has
also been implemented in many clinical microbiology
laboratories for identification and differentiation of both
C. albicans and C. dubliniensis, thus diminishing the
need for germ tube test. However, the production of
light green (for C. albicans) or dark green (for C.
dubliniensis) color is inconsistent when large number of
isolates are screened, thus requiring further cultivation
on a bird-seed agar medium for their accurate
A duplex PCR assay has been developed and extensively
evaluated for the detection and differentiation of clinical
isolates as C. dubliniensis or C. albicans. The method
may be applied directly on clinical yeast isolates for their
identification as C. dubliniensis or C. albicans as it does
not require prior testing for germ tube formation or
other such tests and is suitable for resource-limited
clinical microbiology laboratories equipped with basic PCR
technology. A limitation of the study is that other Candida
and yeast-like organisms will require additional testing for
their species-specific identification.
The authors declare that no competing interests exist.
SA and ZK designed the study, arranged financial support and analyzed the
data. MA, AT and RC performed the experiments. All authors contributed in
writing the manuscript and approved the final version.
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