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Epidemiology of candidemia in Qatar, the Middle East: Performance of

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Epidemiology of candidemia in Qatar,
the Middle East: performance of MALDI-
TOF MS for the identification of Candida
species, species distribution, outcome, and
susceptibility pattern
S. J. Taj-Aldeen, A. Kolecka, R. Boesten,
A. Alolaqi, M. Almaslamani, P. Chandra,
J. F. Meis & T. Boekhout

Infection
A Journal of Infectious Disease

ISSN 0300-8126

Infection
DOI 10.1007/s15010-013-0570-4

1 23
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 Author's personal copy
Infection
DOI 10.1007/s15010-013-0570-4

CLINICAL AND EPIDEMIOLOGICAL STUDY

Epidemiology of candidemia in Qatar, the Middle East:

performance of MALDI-TOF MS for the identification of Candida
species, species distribution, outcome, and susceptibility pattern
S. J. Taj-Aldeen • A. Kolecka • R. Boesten •
A. Alolaqi • M. Almaslamani • P. Chandra •
J. F. Meis • T. Boekhout

Received: 15 August 2013 / Accepted: 2 December 2013

Ó Springer-Verlag Berlin Heidelberg 2013

Abstract of the D1/D2 domains of the large subunit ribosomal DNA

Introduction Bloodstream infections (BSIs) due to Can-
dida spp. constitute the predominant group of hospital-
based fungal infections worldwide. A retrospective study
evaluated the performance of matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) for the identification of BSI Candida
isolates. The epidemiology, risk factors, demographic
features, species distribution, and clinical outcome associ-
ated with candidemia in patients admitted to a single ter-
tiary-care hospital in Qatar, were analyzed.
Methods A single-center, retrospective analysis covering
the period from January 1, 2004 to December 31, 2010 was
performed. Molecular identification used sequence analysis
(LSU rDNA) and the ITS1/2 regions of the rDNA.
MALDI-TOF MS-based identification of all yeast isolates
was performed with the ethanol/formic acid extraction
protocol according to Bruker Daltonics (Bremen, Ger-
many). The susceptibility profiles of 201 isolates to
amphotericin B, itraconazole, fluconazole, voriconazole,
anidulafungin, caspofungin, posaconazole, and isavuco-
nazole were tested using CLSI standard broth microdilu-
tion method (M27-A3 and M27 S4) guidelines. Statistical
analyses were performed with the statistical package SPSS
19.0.
Results A total of 187 patients with 201 episodes of
candidemia were identified. Candida albicans was the most

S. J. Taj-Aldeen (&)
A. Alolaqi J. F. Meis

Mycology Unit, Microbiology Division, Department of
Laboratory Medicine and Pathology, Hamad Medical
Corporation, P.O. Box 3050, Doha, Qatar
e-mail: stajaldeen@hmc.org.qa
A. Kolecka
R. Boesten
T. Boekhout
CBS Fungal Biodiversity Centre, Utrecht, The Netherlands
M. Almaslamani
Infectious Disease Division, Department of Medicine,
Hamad Medical Corporation, Doha, Qatar
M. Almaslamani
Weill Cornell Medical College, Doha, Qatar
P. Chandra
Medical Research Centre, Hamad Medical Corporation,
Doha, Qatar
Department of Medical Microbiology, Radboud University
Medical Center, Nijmegen, The Netherlands
T. Boekhout
Department of Internal Medicine and Infectious Diseases,
University Medical Center, Utrecht, The Netherlands
T. Boekhout
Department of Dermatology, Shanghai Key Laboratory of
Molecular Medical Mycology, Institute of Dermatology and
Medical Mycology, Changzheng Hospital, Second Military
Medical University, Shanghai, People’s Republic of China
T. Boekhout
Institute of Microbiology, Chinese Academy of Sciences,
Beijing, People’s Republic of China

J. F. Meis
Department of Medical Microbiology and Infectious Diseases,
Canisius Wilhelmina Hospital, Nijmegen, The Netherlands

123
 Author's personal copy
common species isolated (33.8 %; n = 68), whereas non-
albicans Candida species represented 66.2 % (n = 133) of
the episodes. The species distribution and outcome of can-
didemia showed a difference in the crude mortality between
patients infected with C. albicans (n = 30; 45.5 %) and non-
albicans Candida species. For example, C. parapsilosis
candidemia was associated with the lowest mortality rate
(40.6 %), and patients with other non-albicans species had
the highest mortality rate (68–71.4 %). High mortality rates
were observed among pediatric (\1 year of age) and elderly
patients ([60 years of age). All strains showed low minimum
inhibitory concentrations (MICs) (MIC90 of 0.063 lg/ml) to
isavuconazole. The overall resistance to voriconazole in vitro
antifungal activity was 2.5 %. C. glabrata (n = 38) had an
MIC90 of 8 lg/ml for fluconazole. Most yeast isolates were
susceptible to anidulafungin ([99.5 %) and 81.1 % to
caspofungin. Resistance to anidulafungin was detected in 1/8
(12.5 %) isolates of C. orthopsilosis. According to new
Clinical and Laboratory Standards Institute (CLSI) break-
points, C. glabrata (n = 38) showed 100 % resistance, and
37/68 (54.4 %) C. albicans isolates were susceptible dose
dependent (SDD) to caspofungin. Identification by MALDI-
TOF MS was in 100 % concordance with molecular
identification.
Conclusion The Middle East epidemiology of candide-
mia has a unique species distribution pattern distinct from
other parts of the globe. High mortality rates were observed
among pediatric (\1 year of age) and elderly patients ([60
years of age). All strains were susceptible to isavuconazole.
All isolates of C.glabrata were resistant to caspofungin
based on M27 S4. MALDI-TOF MS is a highly useful
method for the routine identification of yeast isolates in
clinical setting to achieve successful therapeutic treatment.
Keywords Epidemiology
Candidemia
Bloodstream
infections
Antifungal susceptibility
Risk factors

Outcome
Middle East
MALDI-TOF MS
Introduction
Bloodstream infections due to Candida spp. constitute the
predominant group of hospital-based fungal infections
worldwide [1, 2]. These infections are an important cause of
morbidity and mortality in hospitalized patients with serious
underlying diseases, such as hemato-oncological malignan-
cies, especially immunocompromised patients [3–5]. The
present trend of candidemia shows that a large proportion of
bloodstream infections are due to Candida species other than
C. albicans, particularly among hematological, transplant,
and intensive care unit patients. Fluconazole is often used as
a prophylaxis to prevent invasive yeast infection in patient
populations at high risk, such as those with hemato-
123
S. J. Taj-Aldeen et al.
oncological malignancies and transplant recipients, and it
has also been recommended as a treatment option of Candida
bloodstream infections. The broad use of azole prophylaxis
and therapy has an impact on the incidence of candidemia
and the observed changes in the distribution of the etiologic
agents.
Recent reports of candidemia in a hospital in Germany
identified mortality rates at 30 and 100 days of 65 and
67 %, respectively [5], and this ranged from 44.2 to 61 %
in Brazil [1, 6] and 37–40.4 % in six UK institutes [7, 8].
The crude 12-week adult mortality rate ranged from 34 to
40 % in the USA [9, 10]. Although the global mortality
rate varies greatly between different populations, these data
show that, during the last decade, no significant improve-
ment has been observed and the mortality rate remains
high.
The epidemiology of candidemia has been extensively
studied worldwide, but not in the Middle East. Data on
candidemia in this region are scarce, with local studies
being conducted in a number of medical institutes in the
Gulf region [11, 12]. Important geographical differences
occur worldwide in Candida species distribution and their
patterns of in vitro susceptibilities to antifungal agents.
Thus, performing epidemiological surveillance studies is
important in this part of the world to evaluate potential
changes in species distribution and to assess the antifungal
susceptibility, including that to new agents. The aim of this
retrospective study was to evaluate the performance of
matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS) for the identifica-
tion of bloodstream Candida isolates and to study the
epidemiology, risk factors, demographic features, species
distribution, and clinical outcome associated with candi-
demia in patients admitted to a single tertiary-care hospital,
Hamad Medical Corporation (HMC) in Doha, Qatar.
Materials and methods
Yeast nomenclature
The taxonomic names of yeasts of the species isolated
throughout the present work were based on the most
recently published teleomorphic names (Table 1).
Study design and population
The study was a single-center, retrospective analysis cov-
ering the period from January 1st, 2004 to December 31st,
2010. We analyzed the information pertaining to Candida
bloodstream infections of patients hospitalized in all
departments of HMC, such as hematology/oncology,
pediatric and adult intensive care units, and other medical
 Author's personal copy
Epidemiology of bloodstream candidiasis in Qatar

Table 1 Relationship between genus and species names of anamorphs and teleomorphs of yeasts detected in this study, based on [40]
Teleomorpha Anamorpha Commonly known synonymsa

Unknown Candida albicans (1923)b Candida stellatoidea (1939), Candida albicans var.
stellatoidea (1942)
Unknown Candida dubliniensis (1995)b
Unknown Candida glabrata (1978)b Torulopsis glabrata (1938)
Unknown Candida intermedia (1938)b
Unknown Candida orthopsilosis (2005)b
Unknown Candida parapsilosis (1932)b
Unknown Candida pararugosa (1978)b
Unknown Candida tropicalis (1923)b
Clavispora lusitaniae (1979)b Candida lusitaniae (1959)
Kluyveromyces marxianus (1971)b Candida kefyr (1970)
Cyberlindnera fabianii (2009)b Candida fabianii (1964)b Lindnera fabianii (2008), Hansenula fabianii (1965),
Pichia fabianii (1984)
Lodderomyces elongisporus (1971)b Unknown Saccharomyces elongisporus (1952)
Meyerozyma guilliermondii (2010)b Candida guilliermondii (1938) Pichia guilliermondii (1966)
Pichia kudriavzevii (1965)b Candida krusei (1923) Candida castellanii (1953), Issatchenkia orientalis (1960),
Pichia orientalis (1964)
Wickerhamomyces anomalus (2008)b Candida pelliculosa (1925) Hansenula anomala (1919), Pichia anomala (1984)
Yarrowia lipolytica (1980)b Candida lipolytica (1942)
a
Years of publication are indicated in parentheses
b
Current correct names

wards. The study subject population was composed of all
adult and pediatric hospitalized patients of both genders
who developed candidemia. Candida bloodstream infec-
tions were defined as one or more blood cultures positive
for Candida species in patients with relevant clinical signs
and symptoms [13]. All patients selected for further anal-
yses had at least one positive blood culture of Candida spp.
as identified by the HMC Microbiology Laboratory data-
base. Only the isolate from the first blood culture from each
patient at the time of onset of candidemia was included.
This study was reviewed and approved by the local ethics
committee, Medical Research Center (MRC) at Hamad
Medical Corporation (#11308/11); given the use of retro-
spective laboratory data and preserved yeast species, the
requirement for written informed consent was waived
because of the retrospective and observational nature of
this study.
Biochemical identification
Blood cultures were performed using the Bactec automated
culturing system (BD Diagnostic Systems, Franklin Lakes,
NJ, USA). A small inoculum from a single colony of each
isolate was inoculated onto Sabouraud dextrose agar (SDA)
plates, incubated at 35 °C for 48 h, and used to prepare
inoculum for substrate assimilation profiles employing the
Vitek 2 Compact yeast identification system (bioMérieux,
Marcy-l’Étoile, France), as recommended by the
manufacturer, API ID 32C (bioMérieux), and partly on
CHROMagar Candida plates. Cultures of Candida isolates
were preserved at -70 °C using cryo-tubes (Mast Diag-
nostics, Bootle, Merseyside, UK) until further analysis.
DNA isolation and sequencing
Sequence analysis of the D1/D2 domains of the large
subunit ribosomal DNA (LSU rDNA) and the ITS1 and
ITS2 regions of the rDNA was performed according to the
method of Okoli et al. [14]. The sequences generated were
compared to data available in the NCBI database using the
Basic Local Alignment Search Tool (BLASTn) (http://
blast.ncbi.nlm.nih.gov/).
MALDI-TOF MS
MALDI-TOF MS-based identification of all yeast isolates
was performed according to Bruker Daltonics (Bremen,
Germany) with the ethanol (EtOH)/formic acid (FA)
extraction protocol. For extraction, two loops of yeast
biomass (1-ll volume, sterile inoculation loop) not older
than 24 h growing on SDA were suspended in 300 ll of
molecular graded ionized water and further processed with
900 ll of 95 % EtOH. The volume of FA used for opti-
mization ranged between 20 and 40 ll. However, a volume
of 25 ll of FA was found to be optimal and an equal
volume of acetonitrile (ACN) was added later. From the

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 Author's personal copy
crude protein extract of each tested strain, 1 ll was spotted
in duplicate onto a 96-spot polished steel target plate
(Bruker Daltonics) and allowed to dry at room temperature.
Bacterial test standard (Bruker Daltonics) was also spotted
twice and used as a positive control. Before measurements,
all tested spots were overlaid with 1 ll of alpha-cyano-4-
hydroxycinnamic acid (Bruker Daltonics) saturated matrix
solution. The yeast identification was operated by the
MALDI Biotyper 3.0 system based on mass spectra gen-
erated with the Microflex LT software and compared with
two databases simultaneously. The first was the commer-
cially available database (BDAL) that, at the time of con-
ducting the analyses, contained 3,995 main mass spectra
(MSPs) (Bruker Daltonics), and the second was an CBS-
KNAW in-house library of 510 MSPs of different yeasts
that was established according to the Bruker internal
database creation standard operating procedures.
Identification scores were interpreted according to the
manufacturer’s recommended criteria: a log score value
[2.0 indicated correct identification to the species level, a
log score 1.999 [ value [ 1.7 correct genus recognition,
and no reliable identification with a score \1.7. Each iso-
late was considered correctly identified if at least one of the
duplicates gained scores[2. Strains with results\2.0 or no
peaks found were retested from a fresh culture. The iden-
tification was also considered correct if at least one spot
from the duplicate gave a reliable identification with score
[1.7 that was concordant with the sequencing results.
Susceptibility testing
The susceptibility profiles of the five most commonly iso-
lated species to amphotericin B (AmB) (Bristol-Myers
Squibb, Woerden, The Netherlands), itraconazole (ITC)
(Janssen Research Foundation, Beerse, Belgium), fluco-
nazole (FLC), voriconazole (VOC), anidulafungin (AND)
(Pfizer Central Research, Sandwich, UK), caspofungin
(CSP) (Merck Sharp and Dohme BV, Haarlem, The Neth-
erlands), posaconazole (PSC) (Merck Sharp and Dohme),
and isavuconazole (ISV) (Basilea Pharmaceutica, Basel,
Switzerland, now Astellas) were tested using the standard
broth microdilution method (M27-A3) as recommended by
the Clinical and Laboratory Standards Institute (CLSI) [15].
The susceptibilities were interpreted taking into account the
new species-specific clinical breakpoint, M27-S4 suggested
by the CLSI [16]. The concentrations of AmB, ITC, VOC,
and PSC ranged from 0.016 to 16 lg/ml, that of FLC from
0.063 to 64 mg/l, those of AND and CSP from 0.008 to
8 lg/ml, and that of ISV from 0.004 to 4 lg/ml.
AND and CSP minimal inhibitory concentration (MIC)
cut-off values of B0.25 lg/ml were considered to be sus-
ceptible (S) for C. albicans and C. tropicalis. MIC values
B2 lg/ml were categorized as S for C. parapsilosis and
123
S. J. Taj-Aldeen et al.
Meyerozyma guilliermondii. AND and CSP MIC endpoints
B0.12 lg/ml were considered to be S for C. glabrata. C.
albicans and C. tropicalis strains for which the echino-
candin MIC was C1 lg/ml (C0.5 lg/ml for C. glabrata)
are considered to be resistant (R). C. parapsilosis and M.
guilliermondii strains for which the echinocandin MIC was
C8 lg/ml were considered to be R.
FLC MIC endpoints were categorized as B2 (S), four
susceptible dose-dependent (SDD), and C8 lg/ml (R) for
C. albicans, C. tropicalis, and C. parapsilosis. FLC MIC
values B32 and C64 lg/ml were considered to be SDD and
R, respectively, for C. glabrata. For voriconazole (VOC),
C. albicans, C. tropicalis, and C. parapsilosis were cate-
gorized as MIC B0.12 lg/ml (S), MIC 0.25–0.5 lg/ml
(SDD), and MIC C1 lg/ml. MIC endpoints were deter-
mined for all other Candida species following the M27-S4
CLSI guidelines [16]. The epidemiological cut-off value
(ECV) C1 lg/ml was used to detect VOC resistance in C.
glabrata [17]. A breakpoint B1 lg/ml was selected to
define the isolates as S to AmB [18].
The ranges of MICs and MICs at which 50 and 90 % of
the isolates population of Candida spp. were inhibited
(MIC50 and MIC90, respectively) were calculated. The
MICs of the quality control strains of C. parapsilosis
ATCC 22019 and C. krusei ATCC 6258 were all within the
reference ranges (data not shown).
Statistical analysis
Categorical and continuous values were expressed as fre-
quency (percentage) and mean ± standard deviation (SD).
Descriptive statistics were used to summarize all demo-
graphic and other clinical characteristics of the patients.
Associations between two or more qualitative or categori-
cal variables were assessed using the Chi-square test. For
small cell frequencies, the Chi-square test with continuity
correction factor was used. Quantitative variables means
between more than two independent groups were analyzed
using one-way analysis of variance (ANOVA). Various
diagnostic measures to assess the diagnostic performance
and accuracy of MALDI-TOF MS in the identification of
Candida in reference to molecular technique as a gold
standard, such as sensitivity, specificity, positive, and
negative predictive values, were calculated and reported,
along with their corresponding 95 % confidence limits.
Kappa agreement analysis was performed to assess an
agreement in the identification of Candida between the
MALDI-TOF MS and molecular tests. Pictorial presenta-
tions of the key results were made using appropriate sta-
tistical graphs. A two-sided p-value \0.05 was considered
to be statistically significant. All statistical analyses were
done using the statistical package SPSS 19.0 (SPSS Inc.,
Chicago, IL, USA).
 Author's personal copy
Epidemiology of bloodstream candidiasis in Qatar

Table 2 Demographic clinical characteristics and outcome of candidemia by the most common Candida species
Variables C. albicans, C. glabrata, C. tropicalis, C. orthopsilosis, C. parapsilosis, Others, Total, p-Value
n (%) n (%) n (%) n (%) n (%) n (%) n (%)

Age groupa
\1 year 37 (54.4) 6 (15.8) 9 (25.0) 6 (75.0) 14 (41.2) 6 (35.3) 78 (38.8) 0.0009*
1–18 years 1 (1.5) 2 (5.3) 2 (5.6) 1 (12.5) 5 (14.7) 4 (23.5) 15 (7.5)
19–40 years 2 (2.9) 4 (10.5) 7 (19.4) 0 (0.0) 3 (8.8) 2 (11.8) 18 (9.0)
41–60 years 15 (22.1) 10 (26.3) 3 (8.3) 0 (0.0) 5 (14.5) (0.0) 33 (16.4)
[60 years 13 (19.1) 16 (42.1) 15 (41.7) 1 (12.7) 7 (20.6) 5 (29.4) 57 (28.4)
Total 68 (33.8) 38 (18.9) 36 (17.9) 8 (3.9) 34 (16.9) 17 (8.5) 201 (100)
Genderb
Male 49 (74.2) 22 (66.7) 23 (69.7) 3 (42.9) 17 (53.1) 9 (56.3) 123 (65.8) 0.229
Female 17 (25.8) 11 (33.3) 10 (30.3) 4 (57.1) 15 (46.9) 7 (43.8) 64 (34.2)
Risk categoryb
Heart/ 7 (10.6) 8 (24.2) 9 (27.3) 3 (50.0) 6 (18.8) 5 (31.3) 38 (20.4) 0.153*
pulmonary
Malignancy 14 (21.2) 9 (27.3) 6 (18.2) 0 (0.0) 3 (9.4) 0 (0.0) 32 (17.2)
Renal 6 (9.1) 4 (12.1) 5 (15.2) 0 (0.0) 3 (9.4) 2 (12.5) 20 (10.8)
diseases
Preterm 11 (16.7) 2 (6.1) 1 (3.0) 0 (0.0) 2 (6.3) 1 (6.3) 17 (9.1)
Burn/wound 2 (3.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (6.3) 3 (18.8) 7 (3.8)
Mental 9 (13.6) 3 (9.1) 3 (9.1) 1 (16.7) 2 (6.3) 2 (12.5) 20 (10.8)
disorders
DM 3 (4.5) 3 (9.1) 1 (3.0) 0 (0.0) 0 (0.0) 0 (0.0) 7 (3.8)
GI diseases 2 (3.0) 3 (9.1) 7 (21.2) 1 (16.7) 10 (31.3) 2 (12.5) 25 (13.4)
Others 12 (18.2) 1 (3.0) 1 (3.0) 2 (16.7) 4 (12.5) 1 (6.3) 21 (10.8)
Outcomeb
Alive 36 (54.5) 10 (30.3) 10 (30.3) 2 (28.6) 19 (59.4) 5 (31.3) 82 (43.9) 0.025
Dead 30 (45.5) 23 (69.7) 23 (69.7) 5 (71.4) 13 (40.6) 11 (68.8) 105 (56.1)
* Yates’ corrected Chi-square
a
14 patients had multiple episodes (n = 201)
b
Based on number of subjects (n = 187)

Results (n = 2 each) caused 4 % of infections. Uncommon yeast

Epidemiology
Between the period from January 2004 to December 2010,
a total of 201 Candida isolates were reported from
bloodstream infections of 187 patients at Hamad hospital,
Qatar (14 patients had multiple episodes). The distribution
of Candida species isolated from positive blood cultures
are presented in Table 2. Among the 201 yeast isolates, C.
albicans was the most commonly isolated species (33.8 %;
n = 68), whereas other Candida species comprised 66.2 %
(n = 133) and consisted of C. glabrata (18.9 %; n = 38),
C. tropicalis (17.9 %; n = 36), C. parapsilosis (16.9 %;
n = 34), C. orthopsilosis (3.9 %; n = 8), and C. dublini-
ensis (1.5 %; n = 3). Overall, Pichia kudriavzevii, M.
guilliermondii, Clavispora lusitaniae, and C. pararugosa
species that caused candidemia, such as C. intermedia,
Yarrowia lipolytica, Kluyveromyces lactis, Wickerham-
omyces anomalus, Cyberlindnera fabianii, and Lodder-
omyces elongisporus, were found in single cases each
(3 %). The species distribution and outcome of candidemia
showed a difference in mortality between those patients
infected with C. albicans (n = 30; 45.5 %) and with other
Candida species, which showed a significantly higher
mortality for all other species (68–71.4 %; p = 0.025),
except for C. parapsilosis, which is characterized by a low
mortality ratio (40.6 %) (Table 2; Fig. 1). Patients
belonging to the pediatric age group\1 year of age and the
adult patient age group [60 years of age were most at risk
of candidemia, with high crude mortality rates of 32.4 and
43.8 %, respectively (Fig. 2). The overall crude mortality
of the total population was 56.1 % (Table 2).

123
 Author's personal copy
Fig. 1 Effect of Candida
species on the survival of
patients with candidemia
Fig. 2 Effect of age factor on
the outcome of candidemia
Underlying diseases
In 20 % of the candidemia patients, the underlying disease
was heart/pulmonary disorders, followed by malignancies
(17 % hematological and solid organ tumors), gastroin-
testinal (GI) disease including surgery (13 %), and renal
diseases including transplant patients (11 %). Candidemia
due to other risk factors were between 4 and 11 %. Uni-
variate analysis for risk factors associated with mortality
showed that heart/pulmonary diseases (25/105; 24 %),
malignancies (hematological and solid organ tumors) (23/
105; 22.1 %), GI (11/105; 10.5 %), and renal diseases (13/
105; 12.5 %) were associated with higher mortality.
The age group (Table 2) was found to be significantly
associated with the isolated Candida species (p = 0.0009).
No statistically significant association was observed between
gender and different Candida species (p = 0.229). Overall,
Candida species were the most frequently isolated from
123
S. J. Taj-Aldeen et al.
males (male:female ratio = 123:64). C. albicans (n = 37/
68; 54.4 %), C. parapsilosis (n = 14/34; 41.2 %), and C.
orthopsilosis (n = 6/8; 75 %) predominated in the age group
\1 year. C. glabrata (n = 16/38; 42.1 %) and C. tropicalis
(n = 15/36; 41.7 %) were the predominant yeast species in
patients[60 years old (Table 2). Patients that were below 1
year and over 60 years of age comprised the population most
vulnerable to candidemia, being n = 78/201 (38.8 %) and
n = 57/201 (28.4 %), respectively. Among the total of 93
yeast isolates from pediatric patients (age range 0–18 years),
the species distribution was as follows: C. albicans (n = 38/
68; 55.9 %), C. parapsilosis (n = 19/34; 55.9 %), C. tropi-
calis (n = 11/36; 30.6 %), C. glabrata (n = 8/38; 21.1 %),
C. orthopsilosis (n = 7/8; 87.5 %), and other uncommon
species (n = 10/17; 58.8 %). C. glabrata was frequently
isolated from adult patients over 41 years of age (Table 2).
The following differences (p = 0.153) were found across the
Candida species. C. albicans (14/66; 21.2 %) and C. glabrata
 Author's personal copy
Epidemiology of bloodstream candidiasis in Qatar

Table 3 Susceptibility of the bloodstream Candida species to the Table 3 continued

systemic antifungal agents after 48 h
Range MIC50 Geometric MIC90
Range MIC50 Geometric MIC90 mean
mean
Fluconazole 0.5–1 0.5 0.667 1
All strains (n = 201) Itraconazole \0.016–0.125 0.063 0.035 0.125
Amphotericin B 0.125–2 0.5 0.612 1.0 Voriconazole \0.016–0.063 0.031 0.020 0.063
Fluconazole 0.031–64 0.5 0.332 4 Posaconazole 0.063–0.125 0.125 0.091 0.125
Itraconazole \0.016–16 0.031 0.018 1.00 Isavuconazole \0.016–0.031 \0.016 0.010 0.031
Voriconazole \0.016–1 \0.016 0.013 0.125 Caspofungin 1–2 1 1.143 2
Posaconazole \0.016–2 0.031 0.018 0.500 Anidulafungin 1–4 2 1.524 4
Isavuconazole \0.016–1 \0.016 \0.016 0.063
Caspofungin 0.25–2 0.5 0.498 1
Anidulafungin \0.008–4 0.031 \0.016 2 (9/33; 27.3 %) were the most frequently recovered Candida
C. albicans (n = 68) species associated with malignancies. C. tropicalis was pre-
Amphotericin B 0.25–1 0.5 0.489 1 dominant in patients with heart/pulmonary disorders (9/33;
Fluconazole 0.031–4 0.25 0.161 0.25 27.3 %), and C. parapsilosis was the most common cause of
Itraconazole \0.016–0.25 \0.016 \0.016 \0.016 candidemia in patients with GI diseases (10/32; 31.3 %).
Voriconazole \0.016–0.125 \0.016 \0.016 \0.016
Posaconazole \0.016–0.5 \0.016 \0.016 \0.016 Resistance
Isavuconazole \0.016–0.063 \0.016 \0.016 \0.016
Caspofungin 0.25–0.5 0.5 0.343 0.5 The in vitro susceptibility of 201 isolates to eight anti-
Anidulafungin \0.008–0.016 \0.008 \0.008 \0.008 fungal agents is presented in Table 3. The MIC ranges,
C. tropicalis (n = 36) geometric mean, MIC50, and MIC90 were determined for C.
Amphotericin B 0.5–1 1 0.698 1 albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C.
Fluconazole 0.25–2 0.5 0.460 1 orthopsilosis. Overall, the number of isolates resistant to
Itraconazole \0.016–0.25 0.063 0.026 0.125 the tested antifungals was low. Resistance to ISV was not
Voriconazole \0.016–0.5 0.031 0.023 0.125 detected in any of the Candida strains; this agent exhibited
Posaconazole \0.016–0.5 0.031 0.029 0.25 low MIC values (0.016–1 lg/ml). Resistance to AmB (the
Isavuconazole \0.016–0.25 \0.016 0.010 0.031 MIC cut-off value is B1 lg/ml) was detected in 1/36
Caspofungin 0.25–1 0.5 0.487 1 (2.7 %) isolates C. parapsilosis. CSP exhibited in vitro
Anidulafungin \0.008–0.125 0.063 0.023 0.125 activity against Candida species with MIC 0.25–2 versus
C. parapsilosis (n = 34) \0.008–4 lg/ml for AND. 99.5 % of the isolates tested
Amphotericin B 1–2 1 1.014 1 were susceptible to AND. Resistance to AND was detected
Fluconazole 0.25–4 1 0.709 2 in 1/8 (12.5 %) isolates of C. orthopsilosis. The activity of
Itraconazole \0.016–1 0.063 0.049 0.25 CSP was lower (81.1 % of isolates were susceptible). C.
Voriconazole \0.016–0.125 \0.016 0.011 0.125 glabrata (n = 38) showed 100 % resistance to CSP, and
Posaconazole \0.016–0.5 0.063 0.037 0.125 37/68 (54.4 %) isolates of C. albicans were SDD to CSP.
Isavuconazole \0.016–0.125 0.008 0.009 0.063 All triazoles demonstrated potent activity against C. albi-
Caspofungin 0.5–2 1 1.108 2 cans, C. parapsilosis, and C. orthopsilosis (susceptible rate
Anidulafungin 0.031–4 2 0.365 2 100 %). All isolates of C. glabrata (n = 38; 100 %) were
C. glabrata (n = 38) SDD with FLC, and 3/36 (8.3 %) isolates of C. tropicalis
Amphotericin B 0.5–1 1 0.745 1
were SDD with VOC, whereas resistance to VOC was
Fluconazole 0.5–64 4 2.850 8
detected in 2/38 (5.2 %) isolates of C. glabrata using the
previously reported ECV values [17]. Resistance to itrac-
Itraconazole 0.063 to [16 1 0.438 1
onazole was detected in 22/201 (10.9 %) of the strains, the
Voriconazole \0.016–1 0.125 0.066 0.25
majority of which were C. glabrata.
Posaconazole 0.063–2 0.25 0.279 0.5
Isavuconazole \0.016–1 0.063 0.039 0.25
Yeast identification
Caspofungin 0.5–1 0.5 0.556 1
Anidulafungin \0.008–0.063 0.063 0.034 0.063
Candida isolates routinely tested by biochemical methods
C. orthopsilosis (n = 8)
could not be completely recognized to the species level.
Amphotericin B 1 0.800 1
Twenty-one isolates (10.4 %) were identified to the genus

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Table 4 Species identifications

Species Number of isolates (n = 201)
of Candida and related yeasts
by different diagnostic methods Diagnostic methods
Biochemical, n (%) Molecular, n (%) MALDI-TOF MSa, n (%)

C. albicans 68 (33.8) 68 (33.8) 68 (33.8)

C. glabrata 26 (12.9) 38 (18.9) 38 (18.9)
C. tropicalis 33 (16.4) 36 (17.9) 36 (17.9)
C. orthopsilosis – 8 (4.0) 8 (4.0)
C. parapsilosis 42 (20.9) 34 (16.9) 34 (16.9)
C. dubliniensis 2 (1.0) 3 (1.5) 3 (1.5)
M. guilliermondii – 2 (1.0) 2 (1.0)
a
Candida with species-level P. kudriavzevii 3 (1.5) 2 (1.0) 2 (1.0)
identification (score [2) = 200;
C. lusitaniae 2 (1.0) 2 (1.0) 2 (1.0)
one C. dubliniensis isolate had
identification score (\2) = 1.9 C. pararugosa – 1 (0.5) 1 (0.5)
b
Identified as C. pararugosa C. rugosab 1 (0.5) 1 (0.5) C. pararugosa 1 (0.5) C. pararugosa
by the molecular and MALDI- C. intermedia 1 (0.5) 1 (0.5) 1 (0.5)
TOF MS methods W. anomalus 1 (0.5) 1 (0.5) 1 (0.5)
c
Were identified to the species Y. lipolytica 1 (0.5) 1 (0.5) 1 (0.5)
level by the molecular and
K. lactis – 1 (0.5) 1 (0.5)
MALDI-TOF MS methods (C.
tropicalis, C. glabrata, M. Cy. fabianii – 1 (0.5) 1 (0.5)
guilliermondii, C. pararugosa, L. elongisporus – 1 (0.5) 1 (0.5)
K. lactis, Cy. fabianii, and L. Candida sp.c 21 (10.4) – –
elongisporus)

level only. Other species (total 4.9 %), such as C. ort-
hopsilosis, M. guilliermondii, C. pararugosa, W. anomalus,
Y. lipolytica, K. lactis, Cy. fabianii, and L. elongisporus,
were not identified (Table 4). Two hundred and one iso-
lates were tested by MALDI-TOF MS and yielded scores
that allowed correct species identification. Candida isolates
yielded a high percentage of log score values of C2
(99.5 %). Amongst the Candida species, only one isolate
of C. dubliniensis was correctly identified with a score
value of 1.7 \ value \ 2.0. Considering the sequence
analysis of the ITS regions and D1/D2 domains of the
rDNA as the gold standard for the identification of yeasts,
the MALDI-TOF MS method yields values of diagnostic
accuracy measures such as sensitivity, specificity, positive,
and negative predictive values of 100 %. Kappa agreement
analysis between MALDI-TOF MS and molecular tests
revealed a kappa agreement value for Candida identifica-
tion of 1.00 (i.e., perfect agreement) compared to 0.72 for
the biochemical identification methods.
Discussion
This retrospective candidemia study represents the largest
investigation conducted on Candida-related bloodstream
infections in the Gulf area and reveals, for the first time, the
large burden of candidemia in a main Qatar tertiary care
hospital. In addition, it provides the most representative
and consistent data from the Middle East region on the
epidemiology of candidemia to date [11, 12, 19]. Although
C. albicans remains the most frequently encountered spe-
cies in the clinical laboratory, there has been a worldwide
increase in the frequency of infections caused by non-
albicans Candida species, including C. tropicalis, C. par-
apsilosis, and the intrinsically fluconazole-resistant species
C. glabrata and P. kudriavzevii [9].
The reasons for the emergence of non-albicans Candida
species are not clear, but some medical conditions may
consistently impact the risk of developing candidemia due
to rarely occurring species, other than C. albicans. For
instance C. parapsilosis candidemia has been associated
with vascular catheters and parenteral nutrition [20]. Such
an increase may also be attributed to the improvement at
the diagnostics level, allowing non-albicans Candida spe-
cies to be distinguished with more sensitive methods. In
this study, the medical conditions that consistently
impacted a risk of developing candidemia due to non-
albicans Candida species were as follows: C. glabrata
emerged prominently among patients with malignancies,
C. tropicalis was associated with heart/pulmonary diseases,
and C. parapsilosis was dominant in patients \1 year old
and those with GI diseases.
Although C. glabrata is particularly common in the
northern hemisphere [3], we report a relatively high pro-
portion of C. glabrata (18.9 %) as the second cause of
candidemia in this surveillance analysis. European studies

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Epidemiology of bloodstream candidiasis in Qatar
reported variable proportions of this species in the range
from 9.5 to 19.1 % [21, 22]. A recent study in Italy reported
a change in the epidemiology from 12.8 to 20.8 % [23],
which is close to the proportion reported in the presented
study. Even higher proportions were recently reported in the
UK (33.31 %) [8] and USA (26 %) [9, 24], suggesting a
recent change in the USA epidemiology compared to those
previously reported (12–17 %) [25, 26]. Such a trend is
probably associated with earlier exposure to prophylactic
therapies with fluconazole as a common practice, particu-
larly in patients with malignancies and in intensive care
units [25], and this remains a subject of concern when it
comes to fluconazole resistance. C. glabrata candidemia is
seen more often in older adults and is comparatively less
prevalent in neonates and in the pediatric age group [24].
C. tropicalis has the most prominent geographical varia-
tion over the globe. Interestingly, the Middle East epidemi-
ology of C. tropicalis is different from Western and Eastern
parts of the globe. This species was found to be the third
leading cause of candidemia during this study (17.9 %),
which is close to the values obtained in other Middle Eastern
countries such as Saudi Arabia (19 %) and the United Arab
Emirates (15 %) [12, 19]. Recent US studies reported low
proportions of C. tropicalis candidemia (8.1 to 8.7 %) [9]
and European series have consistently reported the lowest
proportions, namely, the UK (\5 %) [7], Italy (8.2 %) [23],
and Spain (8.21 %) [27]. In contrast, South East Asian
countries reported the highest proportions. In China, the
proportion of C. tropicalis candidemia was 21.8 % [28], in
Thailand 28 % [29], in Taiwan 23.1 % [30], and in India, C.
tropicalis is now the most common cause of hospital-
acquired candidemia. Here, epidemiological studies have
implicated C. tropicalis in up to 45 % of candidemia cases
[31], and such a trend highlights the possibility that this
species may be associated with hospital cross-transmission.
Similar to C. tropicalis, candidemia due to C. parapsi-
losis occurred frequently in the present study, with a
slightly lower proportion (16.9 %) than C. tropicalis. This
high proportion is mainly due to infections in GI patients
(n = 10; 31.3 %) and in the pediatric age group of \1-
year-old patients (n = 14/34; 41.2 %). There is a variation
of C. parapsilosis candidemia in different age groups and
in some regions; C. parapsilosis counts for the majority of
candidemia cases. These findings may be attributed to
transmission through the hands of health care workers [32].
Of the total of 201 episodes of candidemia reported in the
current survey, the absence of C. metapsilosis is of note
and C. orthopsilosis was the fifth most common cause of
candidemia (3.9 %), representing 24 % of the proportion of
C. parapsilosis. In contrast, in China, C. metapsilosis
represented 28.1 % of the C. parapsilosis species complex,
with the absence of C. orthopsilosis. This suggests a geo-
graphical variation in the distribution of species belonging
to the C. parapsilosis species complex [33]. This is the first
report describing C. orthopsilosis as a cause of candidemia
in Qatar. The majority of C. orthopsilosis candidemia cases
(n = 6/8; 75 %) were in the pediatric patients age group
(\1 year of age). C. orthopsilosis has been reported to be
involved in a restricted number of infections [27] and
candidemia caused by this species may be attributed to
their ability to form biofilms [34].
Our study demonstrated that candidemia due to P. ku-
driavzevii is rare in Qatar (\1 %), and that C. glabrata
accounted for the large majority of non-albicans Candida
species. The reason that P. kudriavzevii is rare in Qatar is not
clear, but the wide geographic variability in species distri-
bution suggests that factors such as health care practice,
demographic characteristics, and the use of antibiotics may
be important. Although the proportion of P. kudriavzevii
infections in Qatar is small, the burden of this species is quite
similar to rates recently reported by others [27].
Retrospective cohort studies involving patients with
candidemia and varying underlying diseases and age groups
have revealed worldwide crude mortality rates of 34–67 %
[1, 5–10]. In our study, patients with candidemia had a
crude mortality rate of 56.1 %, which is close to values
reported from Germany and Brazil [1, 5, 6]. Similar to other
reports, patients with C. parapsilosis candidemia had the
lowest death rates [20], while C. glabrata and C. tropicalis
infections were linked to an unfavorable outcome in more
than 40 % of patients [9]. This high crude mortality rate of
candidemia caused by these species may be due to their
occurrence in elderly patients ([60 years old) and in high-
risk category groups, such as patients with cancer. The
severity of the underlying medical condition greatly influ-
ences the crude mortality rate in these patient populations.
In the presented study, the overall susceptibility testing
to antifungal agents revealed the presence of isolates that
were resistant to caspofungin when applying the new
breakpoints (CLSI M27-S4 [16]). All bloodstream isolates
of Candida spp. were susceptible to ISV, as all strains had
low MIC values (\1 lg/ml) and VOC was the azole
showing the best in vitro antifungal activity (97.5 %).
Important differences in susceptibility patterns, especially
for azoles, were observed. High rates of resistant isolates
were only observed for ITC, the majority of which belon-
ged to C. glabrata and, thus, suggesting the possibility of
cross-resistance to other azoles, which leads to treatment
failure. Based on the M27-S4 guideline, FLC was active
against C. albicans, C. tropicalis, and C. parapsilosis, but
have intermediate effect on C. glabrata (n = 38 SDD).
Susceptibility data reported by others were different based
on the M27-A3 document, as FLC resistance rates for
European and North American isolates were 8.1 and 6.6 %,
respectively [27, 35]. Overall, it was observed during this
study that echinocandins exhibited poor activity against C.
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 Author's personal copy
glabrata, C. albicans, and one isolate of C. orthopsilosis.
The high resistance among these species for caspofungin
can be explained by the recently reported variability of
in vitro susceptibility testing [36] and the new CLSI rec-
ommended species-specific breakpoints (CLSI M27-S4
[16]).
AmB was reported as the agent with the lowest overall
resistance rate (0.2 %) [27], and the overall resistance rate
to this agent appeared very low in this study as well.
Although antifungal resistance patterns vary across geo-
graphic regions, the emerging resistance among C. glab-
rata cases in the Middle East region requires further
epidemiological surveillance.
The rank order of species varies worldwide and is
characteristic for each region; moreover, antifungal sus-
ceptibility patterns are species-specific. Consequently, the
rapid and accurate identification of the causative organism
is critical for successful treatment. When growth in blood
culture is detected, yeast cells are usually subcultured on
agar plates to obtain colonies that are used for further
phenotypic testing aimed at the species-level, which typi-
cally takes at least 72 h. When the identification results
based on MALDI-TOF MS, biochemical, and morpholog-
ical features were in concordance, the isolate was identified
as the consensus species. When discordant results were
obtained, definitive species identification was based on
rDNA sequences. Sequence analyses of the D1/D2 domains
and the ITS1/ITS2 regions of the rDNA were performed.
Comparing the performance of each identification tech-
nique, all tested isolates (Table 4) were correctly identified
by MALDI-TOF MS. Species like C. orthopsilosis, M.
guilliermondii, C. pararugosa, K. lactis, Cy. fabianii, and
L. elongisporus were incorrectly identified by phenotypic
methods. Other species such as C. glabrata, C. tropicalis,
C. parapsilosis, C. dubliniensis, and P. kudriavzevii were
correctly identified by routine phenotypic methods in Ha-
mad hospital laboratory (67.7–91.6 %). The overall error in
the identification of clinical isolates by phenotypic methods
was 10.9 %. Thus, contrary to conventional identification
methods, all common yeast isolates were identified by
MALDI-TOF MS in a straightforward fashion. In particu-
lar, C. orthopsilosis, a newly recognized member of the C.
parapsilosis complex that is frequently incorrectly identi-
fied by conventional methods as C. parapsilosis sensu
stricto, and C. pararugosa, which is also difficult to dis-
tinguish by conventional methods, were correctly identified
by MALDI-TOF MS. For the routine clinical diagnostics, it
will be of further interest to consider the MALDI-TOF MS
analyses to identify yeast species and to assess differences
in the antifungal susceptibility profiles of Candida species,
particularly in their resistance to azoles.
MALDI-TOF MS has been reported for the identifica-
tion of yeasts directly from blood cultures [37], but this
123
S. J. Taj-Aldeen et al.
procedure has some limitations, such as the limited diver-
sity of yeast genera and species examined, and it not being
applicable in routine diagnostic laboratories. Instead, we
suggest the culture of yeast isolates onto SDA plates for
24 h before identification by MALDI-TOF MS and when
performed will reduce the turnaround time by 48 h com-
pared to the phenotypic methods. By using the CBS-
KNAW in-house library simultaneously in combination
with the commercial Bruker BDAL database, our data
showed 100 % correct identifications when compared with
molecular analysis as the gold standard (kappa value = 1).
Irrespective of the type and number of tested organisms,
earlier studies showed 96 % (score C1.7) [38] and 97.9 %
[39] correctly identified yeasts by the MALDI-TOF MS
method.
Our study represents, up to now, the largest candidemia
surveillance conducted in the Middle East, Qatar, and
provides data on the species distribution, including less
commonly occurring yeast species, in vitro susceptibility
data of the isolates to eight currently used systemic anti-
fungal agents, and shows the performance of the MALDI-
TOF MS system in the identification of bloodstream-rela-
ted yeast species. Furthermore, this report shows that
candidemia is a significant source of morbidity in Qatar,
with a substantial burden of disease, mortality, and, likely,
high associated costs.
It is important to point out that rapid identification of
yeasts to the species level is necessary to achieve successful
therapeutic treatment. Resistance to antifungal drugs
remains restricted to a few species. However, the Middle
East epidemiology of candidemia has a unique species
distribution pattern, distinct from that of the Eastern and
Western parts of the globe. A prospective epidemiological
surveillance to evaluate the change in candidemia patterns
for the next 4 years in Qatar is underway.
Acknowledgments Supported by Grant NPRP 5-298-3-086 from
the Qatar National Research Fund (a member of Qatar Foundation) to
Saad J. Taj-Aldeen, Muna Almaslamani, Jacques F. Meis, and Teun
Boekhout. The statements herein are solely the responsibility of the
authors.
Conflict of interest J.F.M. received grants from Astellas, Basilea,
and Merck. He has been a consultant to Astellas, Basilea, and Merck
and received speaker’s fees from Merck and Gilead. All other authors
do not report conflicts of interest. All authors contributed to the
content and writing of this manuscript.
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