Increasing prevalence, molecular characterization and antifungal drug susceptibility of serial Candida auris isolates in Kuwait
Increasing prevalence, molecular characterization and antifungal drug susceptibility of serial Candida auris isolates in Kuwait
Ziauddin Khan 0 1 2
Suhail Ahmad 0 1 2
Noura Al-Sweih 0 1 2
Leena Joseph 0 1 2
Wadha Alfouzan 0 1 2
Mohammad Asadzadeh 0 1 2
0 Department of Microbiology, Faculty of Medicine, Kuwait University , Safat , Kuwait
1 University Research Sector Grant No. MI01/15
2 Editor: Alix Therese Coste, Institute of Microbiology , SWITZERLAND
Candida auris is an emerging yeast pathogen of global significance. Its multidrug-resistant nature and inadequacies of conventional identification systems pose diagnostic and therapeutic challenges. This study investigated occurrence of C. auris in clinical specimens in Kuwait and its susceptibility to antifungal agents. Clinical yeast strains isolated during 3.5year period and forming pink-colored colonies on CHROMagar Candida were studied by wet mount examination for microscopic morphology and Vitek 2 yeast identification system. A simple species-specific PCR assay was developed for molecular identification and results were confirmed by PCR-sequencing of rDNA. Antifungal susceptibility testing of one isolate from each patient was determined by Etest. The 280 isolates forming pink-colored colonies on CHROMagar Candida, were identified by Vitek 2 as Candida haemulonii (n = 166), Candida utilis (n = 49), Candida kefyr (n = 45), Candida guilliermondii (n = 9), Candida famata (n = 6) and Candida conglobata (n = 5). Species-specific PCR and PCR-sequencing of rDNA identified 166 C. haemulonii isolates as C. auris (n = 158), C. haemulonii (n = 6) and Candida duobushaemulonii (n = 2). C. auris isolates originated from diverse clinical specimens from 56 patients. Of 56 C. auris isolates tested, all were resistant to fluconazole, 41/ 56 (73%) and 13/56 (23%) were additionally resistant to voriconazole and amphotericin B, respectively. Eleven (20%) isolates were resistant to fluconazole, voriconazole and amphotericin B. One isolate was resistant to caspofungin and micafungin. Increasing isolation of C. auris in recent years from diverse clinical specimens including bloodstream shows that C. auris is an emerging non-albicans Candida species in Kuwait causing a variety of infections. Inability of conventional identification methods to accurately identify this pathogen and multidrug-resistant nature of many strains calls for a greater understanding of its epidemiology, risk factors for acquiring C. auris infection and management strategies in high-risk patients. This is the first comprehensive study on the emergence of this multidrug-resistant yeast from Kuwait and the Middle East.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Competing interests: The authors have declared
that no competing interests exist.
Candida auris is an emerging multidrug-resistant yeast that can cause invasive infections and
is associated with high mortality [1±4]. The species was first identified in 2009 from external
ear sample of a Japanese patient [
]. C. auris has now been recognized as an important
nosocomial pathogen in many countries causing bloodstream infections, ventriculitis, osteomyelitis,
otomastoiditis, chronic ostitis media, intraabdominal infections, pericarditis, pleural efffusion
and vulvovaginitis [1, 3±8]. Outbreaks of C. auris infections have also been reported in
hospitalized patients from Pakistan, the United Kingdom, Spain and Venezuela [2, 9±11]. C. auris is
usually misidentified by routinely used phenotypic methods in clinical microbiology
laboratories and many strains are intrinsically resistant to multiple antifungal agents [
3, 7, 12, 13
to high mortality rates associated with C. auris infections in critically ill patients, this species is
now attracting worldwide attention as an opportunistic fungal pathogen [
3, 7, 14
of molecular identification methods based on sequencing of rDNA and/or protein profiling by
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF
MS) have led to the recognition of C. auris infections in many countries of Africa, Europe,
North and South Americas and Asia including the Middle East [
1, 3, 13, 15
]. Although all
unusual and rare yeast isolates that were referred to the Mycology Reference Laboratory from
2005 were identified by PCR-sequencing of ITS region and/or D1/D2 domains of rDNA, no C.
auris was identified until 2014. However, 7 C. haemulonii strains that caused an outbreak in
Maternity Hospital in Kuwait were identified in 2005±2006 [
]. This retrospective study
investigated the occurrence of C. auris among clinical yeast isolates forming pink-colored
colonies on CHROMagar Candida that were recovered from diverse clinical specimens including
bloodstream during a 3.5-year duration in Kuwait. A simple species-specific PCR assay was
developed for rapid molecular identification of C. auris isolates and the results were confirmed
by PCR-sequencing of rDNA. Antifungal susceptibility testing of one isolate from each patient
was also determined.
Materials and methods
Reference strains, clinical isolates and phenotypic identification
Reference strains or well characterized clinical isolates of Candida dubliniensis (CD36),
Candida albicans (ATCC 90028), Candida glabrata (ATCC 90030), C. parapsilosis (ATCC 22019),
C. orthopsilosis (ATCC 96139), Candida tropicalis (ATCC 750), Candida kefyr (Kw1609/11),
Candida conglobata (Kw381/16), Candida utilis (Kw3642/15), Candida fermentati (Kw3414/
13), Candida guilliermondii (Kw14/14), Candida haemulonii (Kw154/06), Candida
duobushaemulonii (Kw3270/08) and Candida auris (Kw2611/17) were used as reference Candida species.
Clinical yeast strains cultured from various clinical specimens during a 3.5-year (May 2014 to
September 2017) period forming pink-colored colonies on CHROMagar Candida and not
recognized as one of the common Candida spp. by VITEK 2 were subjected to detailed
phenotypic and molecular identification. The clinical specimens (including invasive samples) were
collected from patients after obtaining verbal consent at different hospitals across Kuwait as
part of routine diagnostic work-up for fungal infections. A total of 280 clinical yeast isolates
forming pink-colored colonies on CHROMagar Candida were used. The isolates were sent
from different hospitals across Kuwait to Mycology Reference Laboratory, Department of
Microbiology, Faculty of Medicine; Kuwait University for identification and antifungal
susceptibility testing. The isolates were studied by wet mount examination of the culture for
microscopic morphology and by Vitek 2 yeast identification system (bioMeÂrieux, Marcy-l'Etoile,
France) for species identification. The study was approved by the Committee for the
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Protection of Human Subjects in Research, Health Sciences Center, Kuwait University and
Ministry of Health, Kuwait (Approval no. VDR/EC/2477).
Molecular identification and characterization
The genomic DNA from reference strains and clinical isolates of Candida species was prepared
by the rapid method using Chelex-100, as described previously [
]. A simple, low-cost PCR
assay was developed for rapid molecular identification of C. auris isolates. For this purpose,
one forward (CAURF, 5’-ATTTTGCATACACACTGATTTG-3’) and one reverse (CAURR,
5’-CGTGCAAGCTGTAATTTTGTGA-3’) primer targeting specific sequences within ITS-1
and ITS-2 regions of rDNA of C. auris were synthesized. The unique primer sequences
designed in this study were based on sequence alignment of ITS region sequences from
multiple strains of all commonly encountered clinical yeast species that are available from the
GenBank. The species specificity of the primers CAURF and CAURR for C. auris was indicated by
BLAST searches (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) as they showed complete
sequence identity only with C. auris strains. The reaction and PCR cycling conditions were
same as described previously except that primers CAURF and CAURR were used [
amplicons were run on 2% (w/v) agarose gels, as described previously [
]. The results of
species-specific identification of 16 selected C. auris isolates and all C. haemulonii/C.
duobushaemulonii isolates were confirmed by DNA sequencing of the ITS region of rDNA. The ITS
region was amplified by using ITS1 and ITS4 panfungal primers and both strands of the
amplicons were sequenced by using ITS1FS, ITS2, ITS3 and ITS4RS primers as described previously
]. BLAST searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi?) were performed and >99%
sequence identity was used for species identification.
The genotypic relationship among C. auris was studied by comparing ITS region sequences
of C. auris isolates. Genotypic heterogeneity was further investigated by fingerprinting of the
isolates by using minisatellite-based (M13-MIN, 5’-GAGGGTGGCGGTTCT-3’) and
microsatellite-based (GACA4, 5’-GACAGACAGACAGACA-3’) primers, as described previously
Antifungal susceptibility testing
Antifungal susceptibility of 56 C. auris isolates (representing one isolate from each patient)
was determined by Etest as described previously [
]. Etest strips for amphotericin B (AP),
fluconazole (FL), voriconazole (VO) caspofungin (CS) and micafungin (MYC) (bioMeÂrieux SA,
Marcy-l'-Etoile, France) were used. So far, there are no approved antifungal susceptibility
breakpoints for C. auris. However, based on CLSI and EUCAST susceptibility data, tentative
minimum inhibitory concentration (MIC) breakpoints defining resistance have been
proposed as follows: fluconazole, 32 μg/ml; voriconazole, 1 μg/ml; amphotericin B, 2 μg/ml;
micafungin, 4 μg/ml and caspofungin, 2 μg/ml [
1, 12, 23
Phenotypic and molecular identification of pink-colored colonies on
A total of 280 isolates formed pink-colored colonies on CHROMagar Candida during the
study period and were subjected to detailed phenotypic and molecular characterization. The
Vitek 2 yeast identification system identified these isolates as C. haemulonii (n = 166), Candida
utilis (n = 49), Candida kefyr (n = 45), Candida guilliermondii (n = 9), Candida famata (n = 6)
and Candida conglobata (n = 5) (Table 1).
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aOnly selected isolates for these species were analyzed by PCR-sequencing of rDNA
bAll six isolates identified as C. famata were correctly identified as C. fermentati by PCR-sequencing of rDNA
The PCR amplification performed with CAURF and CAURR primers yielded an expected
size amplicon of nearly 276 bp with DNA extracted from the reference strain of C. auris only
while no amplicon was obtained with genomic DNA prepared from reference strains or well
characterized clinical isolates of C. dubliniensis, C. albicans, C. glabrata, C. parapsilosis, C.
orthopsilosis, C. tropicalis, C. kefyr, C. utilis, C. guilliermondii, C. haemulonii, and C.
duobushaemulonii, as expected (S1 Fig). No amplicon was also obtained with DNA isolated from C.
fermentati as well as with human DNA (data not shown). Of 280 isolates analyzed in this study,
no amplicon was obtained during PCR amplification with CAURF and CAURR primers with
DNA isolated from 114 isolates forming pink-colored colonies on CHROMagar Candida and
identified as C. utilis (n = 49), C. kefyr (n = 45), C. guilliermondii (n = 9), C. famata (n = 6) and
C. conglobata (n = 5) by Vitek 2. However, PCR amplification of DNA isolated from 166
isolates phenotypically identified as C. haemulonii (described above) by Vitek 2 with CAURF and
CAURR primers showed that 158 isolates were actually C. auris as they yielded an amplicon of
276 bp while the remaining 8 isolates did not yield an amplicon (Table 1). PCR-sequencing of
16 selected C. auris isolates matched completely with the ITS region sequence of reference and
several other strains of C. auris from India but were different from the sequences of C. auris
isolates from Japan, South Africa and Venezuela. PCR-sequencing of ITS region of rDNA
also identified the latter 8 isolates as C. haemulonii (n = 6) and C. duobushaemulonii (n = 2)
(Table 1). PCR-sequencing of rDNA also identified all C. utilis, C. conglobata and selected
isolates of C. kefyr and C. guilliermondii. However, all 6 isolates identified as C. famata by Vitek 2
were correctly identified as C. fermentati by PCR-sequencing of rDNA (Table 1). None of the
C. auris isolate showed hyphae or pseudohyphae on wet mount examination of cultures [
Epidemiology of C. auris infections in Kuwait
The 158 C. auris isolates came from 56 (31 males and 25 females) patients (S1 Table). All the
patients yielding C. auris were admitted to intensive care units (ICUs) or other wards in
different hospitals across Kuwait for various life-threatening conditions and for different
time-periods extending up to several months in some cases. The patient's age ranged from 13 to 89
years. None of the C. auris isolate came from a pediatric patient ( 12 years). Thirty-five
patients were Kuwaiti nationals and 10 patients were expatriates originating from 5 different
countries while the remaining 11 were only classified as non-Kuwaiti patients. The origin of
158 C. auris isolates from different specimens is provided in Table 2. Thirty-one patients
yielded multiple isolates either from the same or different clinical specimens and seven
patients remained colonized with C. auris for an extended period of time ranging from 70 days
to 11 months (S1 Table). Interestingly, 16 bloodstream isolates were obtained from 13 patients
and 5 of these patients also yielded C. auris from 1±4 other anatomical sites (S1 Table).
Furthermore, 24 elderly ( 65 year old) patients yielded most (83 of 158, 53%) of the C. auris
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PEG tube swabÐPercutaneous endoscopic gastrostomy tube swab,
Some patients yielded isolates from more than one anatomical site.
isolates described in this study and 21 of these patients were hospitalized in a single healthcare
facility. Also, 12 patients were 80 year old, hospitalized in the same facility and yielded 60
isolates from different/multiple anatomic sites (S1 Table).
Molecular fingerprinting of C. auris isolates
The ITS region of all 16 C. auris isolates that were sequenced were identical. Furthermore,
PCR fingerprinting performed with minisatellite-based (M13-MIN) and microsatellite-based
(GACA4) primers yielded identical patterns of amplified fragments from all C. auris isolates
(data from 6 selected isolates are shown in S2 Fig) suggesting close genotypic relationship
among the isolates.
Antifungal susceptibility testing data
Antifungal susceptibility of C. auris was determined for one representative isolate from each
patient and the data are presented in Table 3. All isolates were resistant to fluconazole with
GM, Geometric mean; SD, standard deviation
Tentative MIC breakpoints: amphotericin B (AP) 2μg/ml; fluconazole (FL) 32μg/ml;
voriconazole (VO) 1 μg/ml; caspofungin (CS) 2μg/ml; micafungin (MYC) 4 μg/ml.
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MICs of 128 to 256 μg/ml. The MICs for voriconazole were quite variable ranging between
0.064 to 6 μg/ml (geometric mean 1.20±1.06 μg/ml). Taking an arbitrary break point of 1 μg/
ml, 41 (73.2%) isolates were additionally resistant to voriconazole. Resistance to amphotericin
B ( 2 μg/ml) was observed in 13 (23.2%) isolates with a geometric mean of 1.05±0.74 μg/ml
(Table 3). Eleven (19.6%) isolates were resistant to fluconazole, voriconazole and amphotericin
B. One isolate obtained from urine specimen of a patient who was operated for colorectal
cancer was resistant to caspofungin and micafungin (MIC = 4 μg/ml). The MIC50 and MIC90
values for amphotericin B, fluconazole, voriconazole, caspofungin and micafungin were 1.5 and
2 μg/ml, 256 and 256 μg/ml, 1.5 and 3 μg/ml, 0.19 and 0.38 μg/ml and 0.094 and 0.125 μg/
ml, respectively (Table 3).
Colony characteristics (pink-colored colonies) of C. auris on CHROMagar Candida are
similar to several other Candida species and commercial yeast identification systems such as
Vitek 2 also routinely identify clinical C. auris isolates as C. haemulonii [
3, 13, 24, 25
the application of molecular methods in clinical microbiology laboratories and some other,
as yet, undefined factors, this multidrug-resistant species is now being isolated from clinical
specimens with increasing frequency worldwide [2, 9±11, 14, 26±32]. C. auris was isolated
for the first time in May 2014 in Kuwait from a case of fungemia in a 27-year-old woman
who was admitted to intensive care unit for septic shock secondary to lobar pneumonia .
During the same year, 4 additional isolates were recovered from urine specimens of three
individual Kuwaiti patients (including 2 isolates from one patient) admitted in the same
hospital. This is despite the fact that all yeast isolates not belonging to common Candida species
were identified by PCR-sequencing of ITS region and/or D1/D2 domains of rDNA from
2005 in Kuwait [34±37]. This approach was successful in identifying 7 C. haemulonii isolates
that caused an outbreak in Maternity hospital in 2005±2006 [
]. The frequency of isolation
of C. auris increased substantially after 2014 as 37 isolates were cultured during 2015, 60
isolates were cultured in 2016 and 56 isolates were cultured until September 2017. Furthermore,
3 and 4 bloodstream isolates were obtained from 2 and 3 candidemia patients in 2015 and
2016, respectively. Consistent with this increasing trend of C. auris infections, 8 bloodstream
isolates from 7 candidemia patients were already recovered in the first 9 months of 2017.
In addition to bloodstream, C. auris was also isolated from tissue specimens obtained from
arteriovenous fistula, abdominal fluid/pleural fluid specimens and eye swab from a case of
endophthalmitis suggesting invasive infections (S1 Table). Interestingly, 24 elderly patients
yielded 83 of 158 (53%) C. auris isolates from different/multiple specimens and were
colonized for extended period of time in some cases. Notably, 21 of 24 elderly patients (including
all 80 year-old patients) with protracted illness were hospitalized in a single healthcare
facility, suggesting the presence/persistence of C. auris in the indoor environment of this
hospital. The persistence of C. auris in the hospital environment has also been noted in
several other studies (7, 9, 10, 31).
All 56 individual patient C. auris isolates were resistant to fluconazole, 13 (23%) were
resistant to fluconazole and amphotericin B and 11 (19.6%) were resistant to fluconazole,
voriconazole and amphotericin B. No obvious reason could be attributed to the sudden increase in the
isolation of this multidrug-resistant pathogen in Kuwait in recent years. Similar observations
of rapid emergence of C. auris in some states of USA, where molecular detection of fungal
pathogens is routinely performed, have also been made [
14, 32, 33
]. Of 174 confirmed clinical
C. auris cases reported from 10 states in USA until November 30, 2017, 148 cases were
reported from only two states on the east coast (New York, n = 110 and New Jersey, n = 38)
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showing a disproportionate distribution of these cases in different states in USA (https://www.
cdc.gov/fungal/diseases/candidiasis/tracking-c-auris.html). The reasons for this
preponderance of C. auris cases in these two states are not clear. Furthermore, 257 patients in only 4
states with clinical cases were found to be colonized with C. auris. A large outbreak of C. auris
infections involving 44 patients has also been reported in hospitalized patients from the United
Kingdom highlighting the rapid emergence and spreading of this multidrug-resistant
pathogen in healthcare facilities in this country [
]. Another large outbreak involving 64 candidemia
cases in Valencia, Spain has also been described recently [Ruiz-Gaitan, unpublished data].
The ability of C. auris isolates to grow at 40±42ÊC, inability to produce hyphae or
pseudohyphae in culture and resistance to fluconazole should prompt clinical microbiology laboratories
to seek identification of the yeast isolate by molecular methods [
3, 24, 25, 38
]. In a recent
study, growth characteristics of four C. auris isolates with other Candida species in Sabouraud
broth and yeast nitrogen base containing 10% NaCl (wt/vol) incubated at 40ÊC with/without
supplementation with dextrose, dulcitol or mannitol were compared . While C. auris
strains grew in this medium, C. haemulonii, C. doubushaemulonii, C. albicans and C.
parapsilosis isolates failed to grow. C. glabrata showed growth only in Sabouraud broth containing
]. Recently, CHROMagar Candida medium supplemented with Pal's medium has
been evaluated for differentiation of C. auris from isolates belonging to C. haemulonii complex
]. However, large-scale evaluation of this medium is required for reliable identification of
For rapid molecular identification, Kordalewska et al. [
] developed a species-specific
thermal PCR and a C. haemulonii complex-specific real-time PCR assay to identify C. auris and
related species (C. haemulonii, C. duobushaemulonii and C. lusitaniae). The species-specific
PCR assay developed in this study for rapid identification of C. auris is also simple and cost
effective as the whole procedure can be completed within 4 hours using basic PCR and gel
electrophoresis equipment and will cost only ~1 US$ per sample (excluding the cost of culture and
personnel time). MALDI-TOF MS can also differentiate C. auris from other Candida species
]. We used 12 other Candida species during specificity testing of our PCR assay. The
DNA from C. fermentati also did not yield an amplicon in PCR, as expected (data not shown).
However, we did not test C. famata, R. glutinis and C. catenulata which are sometimes reported
in place of C. auris by Vitek 2. However, based on BLAST searches, our primers do not show
identity with the sequences of the corresponding region from these species. Although updated
versions of MALDI-TOF MS can identify C. auris, it is yet to be updated in Kuwait.
PCRsequencing of 16 selected C. auris isolates were identical and matched completely with
corresponding sequences of C. auris from India but were different from the sequences of C. auris
isolates from Japan, South Africa and Venezuela. All C. auris isolates also yielded identical
fingerprinting patterns by minisatellite-based and microsatellite-based primers which is
consistent with the highly clonal nature of this yeast species [
2, 7, 27
Consistent with published reports [
2, 3, 24
], all (56, 100%) individual patient's C. auris
isolates were resistant to fluconazole and 41 (73.2%) isolates to voriconazole. Also 13 (23%)
isolates were resistant to amphotericin B highlighting the multidrug-resistant (MDR) nature of
this organism (1±4, 7, 14, 24, 26, 27). Only one isolate in our study was resistant to caspofungin
and micafungin (MIC = 4 μg/ml). This isolate was obtained from the urine specimen of a
patient who was operated for colorectal cancer. Resistance to echinocandins in Candida
species is generally low, not exceeding 3% [
]. Resistance of C. auris to echinocandins has
also been found to be generally lower compared to polyenes [
]. However, resistance in up
to 7% C. auris isolates has been reported in one study [
Although 13 (23%) isolates were classified as resistant to amphotericin B, the actual number
of amphotericin B-resistant isolates may be even higher. This is due to the fact that no clinical
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breakpoints are available for amphotericin B for C. auris. Tentative breakpoints for
fluconazole, amphotericin B, and echinocandins have recently been proposed [
2, 12, 21
]. The Etest
invariably yields much lower MIC values for amphotericin B (presumably due to a narrow
calibration of concentration gradient on the strip). In a previous study comparing MIC
distribution of 90 C. auris isolates by CLSI-BMD, Vitek 2 and Etest, the MIC90 values for amphotericin
B were 4, 16 and 1 μg/ml, respectively [
]. Using tentative breakpoint of 2 μg/ml for
amphotericin B resistance, the percentage of resistant isolates by the CLSI-BMD, Vitek 2 and Etest
were 15.5%, 100% and 1.1%, respectively, indicating considerable differences in the MICs
obtained by the three methods [
]. Based on pharmacokinetic studies in a mouse model of C.
auris infection [
], it has been suggested that isolates showing MIC of 2 μg/ml by broth
microdilution test may be considered as resistant to amphotericin B. Since under standard
dosing, the breakpoints for amphotericin B are estimated to be 1 or 1.5 μg/ml, it may,
therefore, be appropriate if Etest MICs of 1.5 μg/ml for C. auris are also rounded off to 2 μg/ml, as is
practiced for other Candida species [
]. If these revised cut-off values are adopted in our
study, the number of isolates classified as amphotericin B-resistant will climb to 29 (52%).
Candida auris is a persistent colonizer and difficult to eradicate from the hospital
4, 6, 39, 45
]. Its ability to form biofilms on a variety of surfaces and lack of activity of
some antifungal agents such as fluconazole against C. auris may have contributed to its
persistence and nosocomial transmission [
3, 9, 30, 31, 39, 46
]. In our study, two patients (both 80
year-old females) remained colonized for nearly 9 months (P39) and 11 months (P29) in the
respiratory and/or urinary tract and C. auris was also isolated from the catheter tip, however,
both patients did not develop candidemia, likely because they were treated with caspofungin
prophylactically. In addition to demonstrating excellent activity of echinocandins against C.
auris, our data also show that this multidrug-resistant species persists in colonized patients in
hospital settings and the importance of devising effective measures for decontamination of the
patients to prevent further inter- and intra-hospital transmission of C. auris [
3, 4, 6, 47, 48
Our study has a few limitations. i) The MICs for antifungal drugs were only determined by
Etest and not by reference broth microdilution method. ii) The MIC values were determined
for the first isolate from each patient and were not determined for repeat isolates.
Isolation of C. auris from diverse clinical specimens including bloodstream with increasing
frequency in recent years show that C. auris is a rapidly emerging pathogen in Kuwait. A
simple, low-cost PCR assay has also been developed for its rapid identification. All C. auris isolates
obtained from hospitalized patients in Kuwait were resistant to fluconazole, and 13 and 11
isolates were multidrug-resistant strains as they were additionally resistant to amphotericin B
and amphotericin B + voriconazole, respectively. Inability of conventional yeast identification
methods to accurately identify this pathogen and multidrug-resistant nature of many strains
calls for a greater understanding of its epidemiology, risk factors for acquiring C. auris
infection and management strategies in high-risk patients. This is the first comprehensive report
highlighting the increasing prevalence of this multidrug-resistant yeast in Kuwait and the
S1 Table. Source and date of isolation and demographic data of 56 patients yielding 158 C. auris isolates in Kuwait analyzed in this study.
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S1 Fig. Agarose gel of PCR amplicons obtained with C. auris-specific (CAURF and
CAURR) primers and genomic DNA from reference strains of C. dubliniensis (lane 1), C.
albicans (lane 2), C. glabrata (lane 3), C. parapsilosis (lane 3), C. orthopsilosis (lane 4), C.
tropicalis (lane 5), C. kefyr (lane 6), L. conglobata (lane 7), C. utilis (lane 8), C. guilliermondii
(lane 9), C. haemulonii (lane 10), C. duobushaemulonii (lane 11) and C. auris (lane 12).
Lane M is 100 bp DNA ladder and the positions of migration of 100 bp, 300 bp and 600 bp
fragments are marked.
S2 Fig. Agarose gel of PCR products obtained during fingerprinting with minisatellite
based (M13-MIN) primer (Panel A) and microsatellite-based (GACA4) primer (Panel B)
with genomic DNA isolated from 6 C. auris isolates (lanes 1±6). Lane M is 100 bp DNA
ladder and the positions of migration of 100 bp and 600 bp fragments are marked.
We thank Sandhya Vayalil, Soumya Varghese and Omar Al-Musallam for technical assistance.
Conceptualization: Ziauddin Khan, Suhail Ahmad, Noura Al-Sweih.
Data curation: Suhail Ahmad, Leena Joseph, Wadha Alfouzan, Mohammad Asadzadeh.
Formal analysis: Ziauddin Khan, Suhail Ahmad, Leena Joseph, Mohammad Asadzadeh.
Funding acquisition: Ziauddin Khan.
Investigation: Suhail Ahmad, Leena Joseph.
Project administration: Ziauddin Khan.
Resources: Ziauddin Khan, Noura Al-Sweih.
Supervision: Ziauddin Khan, Noura Al-Sweih.
Validation: Wadha Alfouzan.
Visualization: Suhail Ahmad.
Wadha Alfouzan, Mohammad Asadzadeh.
Methodology: Ziauddin Khan, Suhail Ahmad, Leena Joseph, Mohammad Asadzadeh.
Writing ± original draft: Ziauddin Khan, Suhail Ahmad, Noura Al-Sweih, Leena Joseph,
Writing ± review & editing: Ziauddin Khan, Suhail Ahmad, Noura Al-Sweih.
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by Vitek 2, CLSI broth Microdilution, and Etest method. J Clin Microbiol 2015; 53: 1823±1830. https://
doi.org/10.1128/JCM.00367-15 PMID: 25809970
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ERG11 and FKS1 genes in azole and echinocandin resistance. J Antimicrob Chemother 2018 Jan 9.
https://doi.org/10.1093/jac/dkx480 PMID: 29325167
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