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Medical Mycology July 2012, 50, 549555

Microarray technology for yeast identification directly

from positive blood cultures. A multicenter Italian
*Medical Mycology Committee, Italian Society of Clinical Microbiology, Milano, Microbiology Institute , AO Ospedale
San Carlo Borromeo, Milano, Microbiology Laboratory, AOU Maggiore della Carit, Novara, Biochemical and
Microbiology Laboratory, AO Ospedale Civile, Crema, #Microbiology Laboratory, AO Ospedale Domenico Cotugno, Napoli,
^Biochemical and Microbiology Laboratory, PO Ospedale Santo Spirito, Pescara, @Microbiology and Virology Laboratory,
AO Ospedale Niguarda Ca Granda, Milano, $Microbiology Laboratory, AO Ospedale Alessandro Manzoni, Lecco, &
Microbiology and Virology Laboratory, AO Ospedali Riuniti di Ancona, Ancona, %Microbiology Laboratory, IRCCS Policlinico
San Matteo, Pavia, Microbiology Laboratory, AO Ospedali Riuniti di Bergamo, Bergamo, Biochemical and Microbiology
Laboratory, AO Ospedale San Biagio, Alessandria, Microbiology Institute, University of Sassari, Sassari, and
Microbiology Laboratory, AOU Ospedale San Gerardo, Monza, Italy

The authors evaluated the performance of the MycArray Yeast ID (Myconostica Ltd,
UK) assay in the identification of a total of 88 yeast isolates recovered in culture as
compared to that obtained through routine methods. The turn-around time for species
identification directly from cultures by the MycArray was 6 hours, much quicker than
classical methods and all yeasts were correctly identified. In two cases a double identification including Saccharomyces cerevisiae was noted, but it was not confirmed by culture. The results show that MycArray Yeast ID can be a potential tool for rapid detection
and identification of Candida species.
Keywords blood cultures, yeast, identification, microarray

Nosocomial mycoses have become a growing phenomenon over the last decades, and in the USA they increased
more than 207% between the end of the 1970s and the
beginning of the new millennium [1]. This increase is the
result of the growing number of iatrogenic or pathological
immunocompromising conditions, infant prematurity,
solid and hematological cancer, abdominal surgery, presence of medical devices, and the use of broad spectrum
Received 17 August 2011; Received in final revised form 7 November
2011; Accepted 6 December 2011
Correspondence: Claudio Farina, UOC Microbiologia e Virologia AO
Ospedale San Carlo Borromeo, via Pio II no. 3 - 20153 Milano, Italy.
Tel: 39 02402 22456; Fax: 39 02402 22829; E-mail: farina.claudio@

2012 ISHAM

antibiotic therapies [2,3]. In particular, candidemia incidence is 0.51.4/1,000 (in Italy 0.38/1,000) patients/day
and higher in Intensive Care Units at up to 2/1,000
patients/day [3,4].
The current gold standard for detection of candidemia
is culture of blood samples (blood culture). However, this
is not only a time-consuming method, but its sensitivity
for early detection of fungemia has been reported to be
as low as 50% [5]. In order to overcome the limitations
of blood cultures, various molecular approaches for the
identification of pathogenic fungi from blood have been
Molecular techniques are targeted to detect Candida
species in a short period of time, with a high sensitivity and
specificity. Several PCR tests have been devised, such as
nested PCR, multiplex PCR, Taq-man PCR, Light-Cycler
DOI: 10.3109/13693786.2011.648216


Farina et al.

PCR and fluorescent PCR. However, many of these methods are often limited by the number of species that can be
detected in a single assay [610].
The application of a DNA microarray technology may
allow the discrimination of a wide range of pathogens in a
single assay. The aim of the present study was to evaluate
the diagnostic performance of a new microarray method,
MycArray Yeast ID (Myconostica Ltd, Manchester, UK)
and to compare its results with those obtained by culture
considering the advantage of a rapid identification of isolates after their recovery from blood.

Materials and methods

The study was designed to compare the results obtained
with the MycArray Yeast ID to conventional methods for
identifying yeasts recovered in positive blood cultures.
The Medical Mycology Committee (CoSM) of the Italian Society of Clinical Microbiology (AMCLI) collected
data about yeast-positive blood cultures over a 5-month
period (JanuaryMay, 2010). Thirteen hospitals, different
in size and classification, located all over Italy (Northern
Italy: AO Ospedale San Biagio, Alessandria; AO
Ospedali Riuniti, Bergamo; AO Ospedale Civile,
Crema; AO Ospedale Manzoni, Lecco; AO Ospedale
San Carlo Borromeo, Milano; AO Ospedale Niguarda
Ca Granda, Milano; AO Ospedale San Gerardo, Monza;
AOU Maggiore della Carit; Fondazione-IRCSS Policlinico San Matteo, Pavia; Central Italy: AO Ospedali
Riuniti, Ancona; PO Santo Spirito, Pescara; Southern
Italy: AO Ospedale Cotugno, Napoli; Policlinico Universitario, Sassari) participated in the study. All laboratories
were included in a network coordinated by the CoSM
Non-repeat, consecutive positive samples collected at
each participating hospital were submitted to be tested with
the DNA microarray identification system which included
a single positive blood culture for each patient. All culture
bottles (BacT/ALERT; bioMrieux or Bactec; Becton
Dickinson) with visible yeasts presumptively confirmed by
direct Gram staining were sent within three days of recovery to the reference laboratory (AO Ospedale San Carlo
Borromeo, Milano). Once received, they were aliquoted
and stored at both room temperature and 80C, until
confirmation tests and the molecular analysis were performed. Those that could not be processed on the same day
were stored at 80C prior to testing and the length of
storage noted.
The identification of yeast strains was performed at each
hospital according to their routine procedures, mainly by
automated instruments and then confirmed at the reference
laboratory by conventional methods (VITEK2 and ATB ID
32 C; bioMrieux, Marcy lEtoile, France).

A total of 88 Candida spp. were identified using

the standard laboratory procedures and the results were
compared with those obtained by MycArray Yeast ID
(Table 1). Eight negative cultures were included as negative
controls and all tests were performed once. In case of discrepancy in identification, a complete reprocessing of the
samples was performed. In addition, a new subculture was
prepared from the frozen samples and identified by conventional techniques.
DNA was extracted from 1 ml of yeast-positive blood
culture using the MycXtra Fungal DNA kit (UDP2:
and then amplified using the MycArray Yeast ID kit which
specifically amplifies the gene regions encompassing the
probe sequences on the MycArray Yeast ID DNA array
according to the manufacturers instructions. The PCR mix
includes biotinylated dUTP so that the PCR results in biotinylated (labeled) DNA. An independent internal amplification control (IAC) was co-amplified to highlight the
presence of any PCR inhibitor. Thermal cycling parameters
were as follows; initial denaturation at 95C for 10 min;
40 cycles of denaturation at 94C for 15 sec, annealing at
56C for 15 sec, and extension at 72C for 30 sec; and a
final extension at 72C for 5 min.
The Candida species array kit uses the Array Tube
microarray chip technology, i.e., each chip carries probes
designed in the ITS region of the multicopy rRNA complex
of fungi which are specific for species, groups of species,
and probes designed to an exogenous amplification control.
Some probes also detect a number of closely related organisms, i.e., for example one of the Cryptococcus neoformans
probes also detects C. gattii and is expected to detect
C. grubii and Tsuchiyaea wingfieldii. Properties of the used
probes are summarized in Table 1.
Biotin-labeled PCR products were then processed by
MycArray Yeast ID DNA array according to the manufacturers instructions. These were hybridized to the array in
a thermo shaker using a Hybridization Buffer, following
which the probe-target complexes on the chip were washed
with a series of wash buffers. After washing, a blocking
solution was added to the DNA array, and the bound biotinylated DNA molecules were incubated with a Streptavidin-Horseradish Peroxidase (SA-HRP) and then again
washed. A solution of tetramethylbenzidine (TMB), the
SA-HRP-substrate, was added to the DNA array. Bound
SA-HRP converted the TMB into a blue precipitate, resulting in specific spots at the location of the probes to which
target was bound.
The array images were captured using an ArrayTube
Reader with the associated IconoClust software which generated a PDF document with the raw image of the array
and the graphical representation of the array results. Each
probe is present on the array in triplicate and an average
2012 ISHAM, Medical Mycology, 50, 549555

Microarray in yeast identification from blood cultures


Table 1 Species identified by the MycArray Yeast ID kit.


Probe ID

Candida albicans


C. dubliniensis
C. famata
(Debaryomyces hansenii)


C. glabrata


C. guilliermondii


C. krusei
(Issatchenkia orientalis)
C. kefyr
(Kluyveromyces marxianus)


C. parapsilosis group


C. parapsilosis
C. metapsilosis
C. parapsilosis group

C. pelliculosa
C. rugosa
C. tropicalis

C. utilis
Cryptococcus neoformans


Other species detected

D. nepalensis, D. prosopidis,
C. psychrophila, D. maramus,
D. udenii, D. robertsiae,
D. coudertii
D. nepalensis, D. prosopidis,
C. psychrophila
C. caribbica (C. fermentati),
C. fukuyamaensis
(C. xestobii or
C. carpophila)
C. caribbica (C. fermentati),
C. fukuyamaensis (C. xestobii or
C. carpophila), C. smithsonii
Possibly P. cecembensis
Some K. lactis

None outside
C. parapsilosis group

Species with 5 bases

difference or less to probe
(these should not be detected)
C. dubliniensis (3)

D. moranus (2);
D. robertsiae (3);
D. udenii (4)
C. athenensis (2);
C. smithsonii (2)

C. athensensis (2)

K. lactis (4)
K. lactis (3);
P. anomala (5);
S. cerevisiae (5)







Cryptococcus gattii; C. grubii;

C. bacillisporus;
Tsuchiyaea wingfieldii
Cryptococcus gattii;
C. grubii; C. bacillisporus

Filiobasidiella depauperata (4);

Cryptococcus heveanensis (5);
Bullera dendrophila (5)
Tsuchiyaea wingfieldii (3);
Filiobasidiella depauperata (5);
Bullera dendrophila (5)
Coccidioides immitis (2);
Uncinocarpus reesii (2)

Histoplasma capsulatum



Saccharomyces cerevisiae


Saccharomyces sensu stricto:

S. cariocanus, S. paradioxus,
S. mikatae,
S. kudiavzevii, S. boulardii,
S. bayanus,
S. pastorianus, S. uvarum


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Farina et al.

Table 1 (Continued)


Probe ID

Rhodotorula mucilaginosa


R. mucilaginosa (cont.)


Broad-range fungal probes

Internal control



Other species detected

At least some R. toruloides,
R. sphaerocarpum, R. glutinis,
R. dairensis, R. paludigenum,
R. babjevae,
R. kratochvilovae
At least some R. kratochvilovae,
R. araucariae, R. paludigenum,
R. sphaerocarpum, R. glutinis,
R. diobovatum
At least some R. dairensis
One or more of these probes should
pick up all the Candida species given
in the Species column of this table
(including Saccharomyces spp.) as
well as Histoplasma capsulatum and
Rhodotorula spp. These probes will
also detect filamentous fungi
including, Aspergillus spp. and
Coccidioides immitis. The probes
should not pick up Cryptococcus spp.,
bacteria or mammals.

value is used to calculate signal intensity and for organisms

with multiple probes. Most species are represented by multiple probe sequences to capture the known genetic diversity, but not all probes need to be positive to have positive
species identification. Yeast infections due to two or
more species can be also observed. The presence of the
relevant probes was used to confirm the identification
of C. parapsilosis (MA013: 0; MA014: 2; MA0149: 0)
C. metapsilosis (MA013: 1; MA014: 1; MA0149: 0)
and C. orthopsilosis (MA013: 2; MA014: 0; MA0149: 0)
isolates. Thus, for example, MA013 detects C. parapsilosis
well, C. orthopsilosis moderately and C. metapsilosis
poorly. These differences can be exploited for the differentiation of C. parapsilosis group members.
Electronic image and processed data files were electronically stored and species identifications recorded
following the instructions in the IFU. The results from
conventional yeast identification were recorded separately and then compared to the MycArray Yeast ID
data, both in terms of total Candida isolates detected
and final identifications. In cases of discordant species
results, bi-directional sequencing was used as the goldstandard.
For each testing session an internal growth control
was performed which consisted of a 1 ml sample of a
0.5 McFarland suspension of Candida albicans ATCC
90028, C. krusei ATCC 6258, and C. tropicalis ATCC 750

Species with 5 bases

difference or less to probe
(these should not be detected)
R. fluviale (4)




inoculated on a negative blood culture sample to simulate

a real specimen.
The statistical analysis was performed on the grand total
of the isolated strains. Sensitivity and specificity were evaluated. This protocol was approved by the Ethical Committee
at AO Ospedale San Carlo Borromeo, Milano.

The microarray system yielded unequivocal identifications for each of the reference species (Table 2), which
were the same as those obtained by conventional methods. The layout of the chip allows for rapid and accurate
species identification (Fig. 1). Isolates of C. albicans (n
41) were identified by all the three oligonucleotidic
probes (MA001, MA040 and MA041), with the exception of six which were detected only by two probes
(MA040 and MA041). C. parapsilosis (n 10) was similarly identified by three probes (MA013, MA014 and
MA049) and one by two (MA013 and MA049), with
C. glabrata (n 10) detected by three probes (MA011,
MA047 and MA048), C. tropicalis (n 3) by four
(MA004, MA043, MA044 and MA045) but one strain by
two probes (MA043 and MA045) and finally the identification of C. guilliermondii, C. krusei and Cryptococcus
neoformans established by two probes (MA010 and
MA053; MA006 and MA046; MA027 and MA028,
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Microarray in yeast identification from blood cultures


Table 2 Evaluation of the DNA microarray with standard laboratory procedures (VITEK2 and ATB ID 32 C).

Tested species

Positive probe

DNA microarray results

Candida albicans


C. parapsilosis


C. glabrata
C. tropicalis
C. tropicalis
C. lusitaniae
C. guilliermondii
C. krusei
C. neoformans
C. albicans C. glabrata
C. tropicalis C. glabrata


MA001, MA040, MA041

MA040, MA041
MA013, MA014, MA049
MA013, MA049
MA011, MA047, MA048
MA004, MA043, MA044, MA045
MA043, MA045
Not present
MA010, MA053
MA006, MA046
MA027, MA028
MA001, MA040, MA041, MA011, MA047, MA048
MA043, MA045, MA011, MA047, MA048

C. albicans
C. albicans
C. parapsilosis
C. parapsilosis
C. glabrata
C. tropicalis
C. tropicalis
C. guilliermondii
C. krusei
C. neoformans
C. albicans and C. glabrata
C. tropicalis and C. glabrata


isolates identified by standard laboratory procedures.

respectively). In addition, the microarray was found to

discriminate among closely related species, such as
C. orthopsilosis, C. metapsilosis, and C. parapsilosis.
The results obtained with the latter three species indicate
that a level of cross-reactivity still exists with the probes
used in the chip. Nine clinical isolates out of 11, identified as C. parapsilosis, were detected as C. orthopsilosis

and one each as C. metapsilosis and C. parapsilosis by

the microarray system. Mixed infections (C. albicans and
C. glabrata, n 7; C. tropicalis and C. glabrata, n 1)
were correctly identified.
However, two cases caused by C. lusitaniae were
not identified as expected, and in two cases, a double
identification including Saccharomyces cerevisiae was

Fig. 1 MycArray Yeast ID results. Column 1 shows the micro array profile. Column 2 shows the PDF output with the graphical representation of results.
Signal strengths (Y-axis) are normalized against the biotin marker controls. The number of each probe is shown below the X-axis and the organism is
named above each column. In this example, all the control reactions were successful and C. parapsilosis was identified. Lines: (A) Cryptococcus
neoformans; (B) C. albicans.

2012 ISHAM, Medical Mycology, 50, 549555


Farina et al.

different from those obtained by conventional methods

(only C. albicans and C. tropicalis, respectively). The conventional identification was therefore repeated which confirmed that the array identification was incorrect.
The agreement between the traditional and the microarray techniques was 95.5% (84/88: 8 negative and 76
positive cultures). Complete agreement was observed for
all the species included in the microarray pattern with
the test negative in two instances in which the yeasts
recognized by Gram staining and culture but not included
in the microarray pattern (C. lusitaniae, 2).
Finally, the technical sensitivity and specificity of the
MycArray Yeast ID DNA array evaluated for the identified
yeast species was 100%, and 99.8%, respectively. However, its sensitivity and specificity for the blood cultures
samples was 100%, and 80%, respectively.

Fungemia represents the 4th most common cause of bloodstream infections (BSI) in hospitalized patients in the USA
and the 7th in Europe [11,12]. It is related to longer hospitalizations and overall candidemia accounts for a 3575%
mortality rate [13]. It is well documented that its prognosis
is strictly related to the initiation of therapy, and that mortality increases more than 25% if there are delays between
the blood collection and the report of Candida recovery of
more than 48 hours [13].
The most common etiologic agent is C. albicans, followed by C. glabrata, C. parapsilosis, C. tropicalis and
C. krusei, with these five species accounting for more than
90% of all human cases.
Several other fungi can also cause fungemia including
Cryptococcus spp., S. cerevisiae, Histoplasma capsulatum,
Rhodotorula spp. and other Candida species. In Italy, many
studies suggest that C. parapsilosis is second to C. albicans
as the most common species recovered, being much more
frequent than all other Candida species [14].
Determining the etiology is important in the selection
of appropriate antifungal therapy because non-C. albicans
Candida species are more frequent and generally more
resistant to common therapies. For example, C. krusei is
fluconazole resistant and Rhodotorula spp., H. capsulatum
and Cryptococcus spp. are resistant to echinocandins [15].
The identification of the yeasts isolated from blood
cultures to the species level has become more and more
important to assure the best clinical management of
fungemia patients. In fact, the clinical outcome is related
to the early initiation of therapy, and the timely administration of antifungal drugs [1618].
Differences in virulence and in antifungal drug susceptibility of Candida spp. make identification and MIC
determination very important for clinical management

[19,20]. The conventional method of identification of

pathogenic fungi used in clinical microbiology is too
time-consuming to manage patients [21]. The application
of DNA microarray technology, which may enable discrimination of a wide range of pathogens in a single assay,
would improve patient outcomes.
In this study, the turn-around time from recovery from
blood samples to identification with the MycArray Yeast ID
was 6 h, much quicker than could be achieved with classical isolation (1872 h) and identification (848 h) methods.
In addition, species identification can be determined even
if dedicated personnel are present in the laboratory.
The detection of fungal pathogens involved in invasive
mycoses using the microarray technology has been already
been assessed in 2007 from Spiess et al. [22]. Therefore,
the MycArray Yeast ID technique to be considered as a
diagnostic tool for the routine clinical laboratory should be
implemented to identify a higher number of clinically relevant species and to do so in clinical samples.
The MycArray Yeast ID technology is a very promising
and rapid tool for the identification of recovered potential
pathogens but cannot be used for their direct detection in
clinical samples. It can bring an important improvement to
the standard identification procedure, even if it is now only
in research facilities.
Furthermore, the MycArray Yeast ID technology, combined with the possibility to detect virulence factors or
genes that confer resistance to antifungals, would lead to
more efficient therapies.

This study was supported by the financial support of
Myconostica Ltd, UK, (grant number for financial aid: 231
Declaration of interest: The authors report no conflicts of
interest. The authors alone are responsible for the content
and writing of the paper.

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