Increasing prevalence, molecular characterization and antifungal drug susceptibility of serial Candida auris isolates in Kuwait

PLOS ONE, Apr 2018

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 3.5-year 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.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0195743&type=printable

Increasing prevalence, molecular characterization and antifungal drug susceptibility of serial Candida auris isolates in Kuwait

April 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 files. Competing interests: The authors have declared that no competing interests exist. Introduction 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 [ 5 ]. 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 ]. Due 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 ]. Application 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 [ 16 ]. 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 2 / 12 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 [ 17 ]. 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 [ 17 ]. PCR amplicons were run on 2% (w/v) agarose gels, as described previously [ 18 ]. 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 [ 19, 20 ]. 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 [ 21 ]. Antifungal susceptibility testing Antifungal susceptibility of 56 C. auris isolates (representing one isolate from each patient) was determined by Etest as described previously [ 22 ]. 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 ]. Results Phenotypic and molecular identification of pink-colored colonies on CHROMagar Candida 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). 3 / 12 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 [ 3 ]. 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 4 / 12 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. 5 / 12 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). Discussion 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 ]. With 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 [33]. 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 [ 16 ]. 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) 6 / 12 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 [ 9 ]. 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 [39]. 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 dextrose [ 39 ]. Recently, CHROMagar Candida medium supplemented with Pal's medium has been evaluated for differentiation of C. auris from isolates belonging to C. haemulonii complex [ 40 ]. However, large-scale evaluation of this medium is required for reliable identification of C. auris. For rapid molecular identification, Kordalewska et al. [ 15 ] 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 [ 24, 25 ]. 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% [ 41, 42 ]. Resistance of C. auris to echinocandins has also been found to be generally lower compared to polyenes [ 3, 27 ]. However, resistance in up to 7% C. auris isolates has been reported in one study [ 24 ]. 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 7 / 12 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 [ 24 ]. 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 [ 24 ]. Based on pharmacokinetic studies in a mouse model of C. auris infection [ 43 ], 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 [ 21, 44 ]. 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 environment [ 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. Conclusions 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 Middle East. Supporting information S1 Table. Source and date of isolation and demographic data of 56 patients yielding 158 C. auris isolates in Kuwait analyzed in this study. (DOCX) 8 / 12 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. (DOCX) (DOCX) Acknowledgments 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. Author Contributions 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. 9 / 12 10 / 12 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 11 / 12 ERG11 and FKS1 genes in azole and echinocandin resistance. J Antimicrob Chemother 2018 Jan 9. https://doi.org/10.1093/jac/dkx480 PMID: 29325167 1. Sears D , Schwartz BS . Candida auris: An emerging multidrug-resistant pathogen . Int J Infect Dis 2017 ; 63 : 95 ± 98 . https://doi.org/10.1016/j.ijid. 2017 . 08 .017 PMID: 28888662 2. Lockhart SR , Etienne KA , Vallabhaneni S , Farooqi J , Chowdhary A , Govender NP , et al. Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses . Clin Infect Dis 2017 ; 64 : 134 ± 140 . https://doi.org/10.1093/cid/ ciw691 PMID: 27988485 3. Khan Z , Ahmad S. Candida auris: an emerging multidrug-resistant pathogen of global significance . Curr Med Res Pract 2017 ; 7 : 240 ± 248 . 4. Jeffery-Smith A , Taori SK , Schelenz S , Jeffery K , Johnson EM , Borman A , et al. Candida auris: a review of the literature . Clin Microbiol Infect 2018 ; 31 : e00029± 17 . 5. Satoh K , Makimura K , Hasumi Y , Nishiyama Y , Uchida K , Yamaguchi H . Candida auris sp. nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital . Microbiol Immunol 2009 ; 53 : 41 ± 44 . https://doi.org/10.1111/j.1348- 0421 . 2008 . 00083 . x PMID : 19161556 6. Sipavak ES , Hanson KE . Candida auris: An emerging fungal pathogen . J Clin Microbiol Online 22 November 2017 . 7. Chowdhary A , Sharma C , Meis JF . Candida auris: A rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally . PLoS Pathog 2017 ; 13 : e1006290. https://doi.org/10.1371/ journal.ppat. 1006290 PMID: 28542486 8. Morales-LoÂpez SE , Parra-Giraldo CM , Ceballos-GarzoÂn A , MartÂõnez HP , RodrÂõguez GJ , AÂlvarezMoreno CA , et al. Invasive infections with multidrug-resistant yeast Candida auris , Colombia. Emerg Infect Dis 2017 ; 23 : 162 ± 164 . https://doi.org/10.3201/eid2301.161497 PMID: 27983941 9. Schelenz S , Hagen F , Rhodes JL , Abdolrasouli A , Chowdhary A , Hall A , et al. First hospital outbreak of the globally emerging Candida auris in a European hospital . Antimicrob Resist Infect Control 2016 ; 5 : 35 . https://doi.org/10.1186/s13756-016 -0132-5 PMID: 27777756 10. Calvo B , Melo AS , Perozo-Mena A , Hernandez M , Francisco EC , Hagen F , et al. First report of Candida auris in America: Clinical and microbiological aspects of 18 episodes of candidemia . J Infect 2016 ; 73 : 369 ± 374 . https://doi.org/10.1016/j.jinf. 2016 . 07 .008 PMID: 27452195 11. Ruiz GaitaÂn AC , Moret A , LoÂpez Hontangas JL , Molina JM , Aleixandre LoÂpez AI , Cabezas AH , et al. Nosocomial fungemia by Candida auris: first four reported cases in continental Europe . Rev Iberoam Micol 2017 ; 34 : 23 ± 27 . https://doi.org/10.1016/j.riam. 2016 . 11 .002 PMID: 28131716 12. Arendrup MC , Prakash A , Meletiadis J , Sharma C , Chowdhary A. Comparison of EUCAST and CLSI reference microdilution MICs of eight antifungal compounds for Candida auris and associated tentative epidemiological cutoff values . Antimicrob Agents Chemother 2017 ; 61 . 13. Lockhart SR , Jackson BR , Vallabhaneni S , Ostrosky-Zeichner L , Pappas PG , Chiller T. Thinking beyond the common Candida species: need for speciation of Candida due to the emergence of multidrug resistant Candida auris . J Clin Microbiol 2017 ; 55 : 3324 ± 3327 . https://doi.org/10.1128/JCM. 01355-17 PMID: 28904185 14. Tsay S , Kallen A , Jackson BR , Chiller TM , Vallabhaneni S. Approach to the investigation and management of patients with Candida auris, an emerging multidrug-resistant yeast . Clin Infect Dis 2017 Aug 17 . https://doi.org/10.1093/cid/cix744 [Epub ahead of print]. PMID: 29020224 15. Kordalewska M , Zhao Y , Lockhart SR , Chowdhary A , Berrio I , Perlin DS . Rapid and accurate molecular identification of the emerging multidrug-resistant pathogen Candida auris . J Clin Microbiol 2017 ; 55 : 2445 ± 2452 . https://doi.org/10.1128/JCM.00630-17 PMID: 28539346 16. Khan ZU , Al-Sweih NA , Ahmad S , Al-Kazemi N , Khan S , Joseph L , Chandy R . Outbreak of fungemia among neonates caused by Candida haemulonii resistant to amphotericin B, itraconazole, and fluconazole . J Clin Microbiol 2007 ; 45: 2025 ± 2027 . https://doi.org/10.1128/JCM.00222-07 PMID: 17428940 17. Asadzadeh M , Ahmad S , Hagen F , Meis JF , Al-Sweih N , Khan Z. Simple, low-cost detection of Candida parapsilosis complex isolates and molecular fingerprinting of Candida orthopsilosis strains in Kuwait by ITS region sequencing and amplified fragment length polymorphism analysis . PLoS One 2015 ; 10 : e0142880. https://doi.org/10.1371/journal.pone. 0142880 PMID: 26580965 18. Ahmad S , Mustafa AS , Khan Z , Al-Rifaiy AI , Khan ZU . PCR-enzyme immunoassay of rDNA in the diagnosis of candidemia and comparison with amplicon detection by agarose gel electrophoresis . Int J Med Microbiol 2004 ; 294 : 45 ± 51 . https://doi.org/10.1016/j.ijmm. 2004 . 01 .002 PMID: 15293453 19. Khan ZU , Ahmad S , Mokaddas E , Chandy R , Cano J , Guarro J . Actinomucor elegans var. kuwaitiensis isolated from the wound of a diabetic patient . Antonie van Leeuwoenhoek 2008 ; 94 : 343 ± 352 . 20. Khan ZU , Ahmad S , Hagen F , Fell JW , Kowshik T , Chandy R , et al. Cryptococcus randhawai sp. nov., a novel anamorphic basidiomycetous yeast isolated from tree trunk hollow of Ficus religiosa (peepal tree) from New Delhi, India . Antonie Van Leeuwenhoek 2010 ; 97 : 253 ± 259 . https://doi.org/10.1007/s10482- 009 -9406-8 PMID: 20091225 21. Ahmad S , Khan Z , Mustafa AS , Khan ZU . Epidemiology of Candida colonization in an intensive care unit of a teaching hospital in Kuwait . Med Mycol 2003 ; 41 : 487 ± 493 . PMID: 14725322 22. Asadzadeh M , Al-Sweih NA , Ahmad S , Khan ZU . Antifungal susceptibility of clinical Candida parapsilosis isolates in Kuwait . Mycoses 2008 ; 51 : 318 ± 323 . https://doi.org/10.1111/j.1439- 0507 . 2008 . 01492 . x PMID : 18855844 23. CDC. Recommendations for identification of Candida auris . https://www.cdc.gov/fungal/diseases/ candidiasis/recommendations.html. 2017 . 24. Kathuria S , Singh PK , Sharma C , Prakash A , Masih A , Kumar A , et al. Multidrug-resistant Candida auris misidentified as Candida haemulonii: characterization by matrix-assisted laser desorption ionizationtime of flight mass spectrometry and DNA sequencing and its antifungal susceptibility profile variability 25. Mizusawa M , Miller H , Green R , Lee R , Durante M , Perkins R et al. Can a multi-drug resistant Candida auris be reliably identified in clinical microbiology laboratories ? J Clin Microbiol 2017 ; 55 : 638 ± 640 . https://doi.org/10.1128/JCM.02202-16 PMID: 27881617 26. Chowdhary A , Anil Kumar V , Sharma C , Prakash A , Agarwal K , Babu R , et al. Multidrug-resistant endemic clonal strain of Candida auris in India . Eur J Clin Microbiol Infect Dis 2014 ; 33 : 919 ± 926 . https://doi.org/10.1007/s10096-013 -2027-1 PMID: 24357342 27. Chowdhary A , Prakash A , Sharma C , Kordalewska M , Kumar A , Sarma S , et al. A multicentre study of antifungal susceptibility patterns among 350 Candida auris isolates ( 2009 ± 17) in India: role of ERG11 and FKS1 genes in azole and echinocandin resistance . J Antimicrob Chemother 2017 https://doi.org/ 10.1093/jac/dkx480 PMID: 29325167 28. Magobo RE , Corcoran C , Seetharam S , Govender NP . Candida auris-associated candidemia, South Africa . Emerg Infect Dis 2014 ; 20 : 1250 ± 1251 . https://doi.org/10.3201/eid2007.131765 PMID: 24963796 29. Chakrabarti A , Sood P , Rudramurthy SM , Chen S , Kaur H , Capoor M , et al. Incidence, characteristics and outcome of ICU-acquired candidemia in India . Intensive Care Med 2015 ; 41 : 285 ± 295 . https://doi. org/10.1007/s00134-014 -3603-2 PMID: 25510301 30. Rudramurthy SM , Chakrabarti A , Paul RA , Sood P , Kaur H , Capoor MR , et al. Candida auris candidaemia in Indian ICUs: analysis of risk factors . J Antimicrob Chemother 2017 ; 72 : 1794 ± 1801 . https://doi. org/10.1093/jac/dkx034 PMID: 28333181 31. Vallabhaneni S , Kallen A , Tsay S , Chow N , Welsh R , Kerins J , et al. Investigation of the first seven reported cases of Candida auris, a globally emerging invasive, multidrug-resistant fungusÐUnited States , May 2013- August 2016 . MMWR Morb Mortal Wkly Rep 2016 ; 65 : 1234 ± 1237 . https://doi.org/ 10.15585/mmwr.mm6544e1 PMID: 27832049 32. Vallabhaneni S , Kallen A , Tsay S , Chow N , Welsh R , Kerins J , et al. Investigation of the First Seven Reported Cases of Candida auris, a Globally Emerging Invasive , Multidrug-Resistant Fungus-United States , May 2013- August 2016 . Am J Transplant 2017 ; 17 : 296 ± 299 . https://doi.org/10.1111/ajt.14121 PMID: 28029734 33. Emara M , Ahmad S , Khan Z , Joseph L , Al-Obaid I , Purohit P , et al. Candida auris candidemia in Kuwait , 2014 . Emerg Infect Dis 2015 ; 21 : 1091 ± 1092 . https://doi.org/10.3201/eid2106.150270 PMID: 25989098 34. Al-Sweih N , Khan ZU , Ahmad S , Devarajan L , Khan S , Joseph L , et al. Kodamaea ohmeri as an emerging pathogen: a case report and review of the literature . Med Mycol 2011 ; 49 : 766 ± 770 . https://doi.org/ 10.3109/13693786. 2011 .572300 PMID: 21438792 35. Al-Sweih N , Ahmad S , Joseph L , Khan S , Khan Z. Malassezia pachydermatis fungemia in a preterm neonate resistant to fluconazole and flucytosine . Med Mycol Case Rep 2014 ; 5 : 9± 11 . https://doi.org/ 10.1016/j.mmcr. 2014 . 04 .004 PMID: 24936403 36. Al-Sweih N , Ahmad S , Joseph L , Khan S , Vayalil S , Chandy R , et al. Candida fermentati as a cause of persistent fungemia in a preterm neonate successfully treated by combination therapy with amphotericin B and caspofungin . J Clin Microbiol 2015 ; 53 : 1038 ± 1041 . https://doi.org/10.1128/JCM.03351-14 PMID: 25568433 37. Al-Sweih N , Ahmad S , Khan S , Khan Z , Joseph L , Vayalil S , et al. Persistent Candida conglobata bloodstream infection in a preterm neonate successfully treated by combination therapy with amphotericin B and caspofungin . J Mycol Med 2017 ; 27 : 271 ± 276 . https://doi.org/10.1016/j.mycmed. 2017 . 01 .010 PMID: 28189376 38. Ben-Ami R , Berman J , Novikov A , Bash E , Shachor-Meyouhas Y , Zakin S , et al. Multidrug-resistant Candida haemulonii and C. auris , Tel Aviv, Israel. Emerg Infect Dis 2017 ; 23 : 195 ± 203 . 39. Welsh RM , Bentz ML , Shams A , Houston H , Lyons A , Rose LJ , et al. Survival, persistence, and isolation of the emerging multidrug-resistant pathogenic yeast Candida auris on a plastic health care surface . J Clin Microbiol 2017 ; 55 : 2996 ± 3005 . https://doi.org/10.1128/JCM.00921-17 PMID: 28747370 40. Kumar A , Sachu A , Mohan K , Vinod V , Dinesh K , Karim S. Simple low cost differentiation of Candida auris from Candida haemulonii complex using CHROMagar Candida medium supplemented with Pal's medium . Rev Iberoam Micol 2017 ; 34 : 109 ± 111 . https://doi.org/10.1016/j.riam. 2016 . 11 .004 PMID: 28392225 41. Castanheira M , Messer SA , Rhomberg PR , Pfaller MA . Antifungal susceptibility patterns of a global collection of fungal isolates: results of the SENTRY Antifungal Surveillance Program ( 2013 ). Diagn Microbiol Infect Dis 2016 ; 85 : 200 ± 204 . https://doi.org/10.1016/j.diagmicrobio. 2016 . 02 .009 PMID: 27061369 42. Chowdhary A , Prakash A , Sharma C , Kordalewska M , Kumar A , Sarma S , et al. A multicentre study of antifungal susceptibility patterns among 350 Candida auris isolates ( 2009 ± 17) in India: role of the 43. Lepak Alexander J , Miao Zhao , Berkow Elizabeth L , Lockhart Shawn R , Andes David R. Pharmacodynamic optimization for treatment of invasive Candida auris infection . Antimicrob Agents Chemother 2017 , http://dx.doi.org/10.1128/AAC.00791- 17 . 44. Lass-FloÈrl C , Arendrup MC , Rodriguez-Tudela JL , Cuenca-Estrella M , Donnelly P , Hope W ; European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing . EUCAST technical note on Amphotericin B. Clin Microbiol Infect 2011 ; 17 : E27 ± 29 . https://doi.org/10. 1111/j.1469- 0691 . 2011 . 03644 . x PMID : 22011310 45. Lesho EP , Bronstein MZ , McGann P , Stam J , Kwak Y , Maybank R , et al. Importation, mitigation, and genomic epidemiology of Candida auris at a large teaching hospital . Infect Control Hosp Epidemiol 2017 Dec 6 : 1 ±5. https://doi.org/10.1017/ice. 2017 .231 PMID: 29208056 46. Kean R , Sherry L , Townsend E , McKloud E , Short B , Akinbobola A , et al. Surface disinfection challenges for Candida auris: an in vitro study . J Hosp Infect. 2017 Dec 1 . https://doi.org/10.1016/j.jhin. 2017 . 11 .015 [Epub ahead of print]. PMID: 29203448 47. Abdolrasouli A , Armstrong-James D , Ryan L , Schelenz S. In vitro efficacy of disinfectants utilised for skin decolonisation and environmental decontamination during a hospital outbreak with Candida auris . Mycoses 2017 Sep 5 . https://doi.org/10.1111/myc.12699 [Epub ahead of print]. PMID: 28872735 48. Cadnum JL , Shaikh AA , Piedrahita CT , Sankar T , Jencson AL , Larkin EL , et al. Effectiveness of disinfectants against Candida auris and other Candida species . Infect Control Hosp Epidemiol 2017 ; 38 : 1240 ± 1243 . https://doi.org/10.1017/ice. 2017 .162 PMID: 28793937


This is a preview of a remote PDF: http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0195743&type=printable

Ziauddin Khan, Suhail Ahmad, Noura Al-Sweih, Leena Joseph, Wadha Alfouzan, Mohammad Asadzadeh. Increasing prevalence, molecular characterization and antifungal drug susceptibility of serial Candida auris isolates in Kuwait, PLOS ONE, 2018, DOI: 10.1371/journal.pone.0195743