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The molecular basis of reduced susceptibility to amphotericin B (rs-AMB) among any yeasts is poorly defined. Genetic alterations in genes involved in ergosterol biosynthesis and total cell sterols were investigated among clinical Candida kefyr isolates. C. kefyr isolates ( n  = 81) obtained from 74 patients in Kuwait and identified by phenotypic and molecular methods were analyzed. An Etest was initially used to identify isolates with rs-AMB. Specific mutations in ERG2 and ERG6 involved in ergosterol biosynthesis were detected by PCR sequencing. Twelve selected isolates were also tested by the SensiTitre Yeast One (SYO), and total cell sterols were evaluated by gas chromatography-mass spectrometry and ERG3 and ERG11 sequencing. Eight isolates from 8 patients showed rs-AMB by Etest, including 2 isolates with additional resistance to fluconazole or to all three antifungals. SYO correctly identified 8 of 8 rs-AMB isolates. A nonsynonymous mutation in ERG2 was detected in 6 of 8 rs-AMB isolates but also in 3 of 73 isolates with a wild-type AMB pattern. One rs-AMB isolate contained a deletion (frameshift) mutation in ERG2 . One or more nonsynonymous mutations was detected in ERG6 in 11 of 81 isolates with the rs-AMB or wild-type AMB pattern. Among 12 selected isolates, 2 and 2 isolates contained a nonsynonymous mutation(s) in ERG3 and ERG11 , respectively. Ergosterol was undetectable in 7 of 8 rs-AMB isolates, and the total cell sterol profiles were consistent with loss of ERG2 function in 6 rs-AMB isolates and loss of ERG3 activity in another rs-AMB isolate. Our data showed that ERG2 is a major target conferring rs-AMB in clinical C. kefyr isolates. IMPORTANCE Some yeast species exhibit intrinsic resistance or rapidly acquire resistance to azole antifungals. Despite >50 years of clinical use, resistance to amphotericin B (AMB) among yeast species has been extremely rarely reported until recently. Reduced susceptibility to AMB (rs-AMB) among yeast species is, therefore, a matter of serious concern due to the availability of only four classes of antifungal drugs. Recent studies in Candida glabrata, Candida lusitaniae, and Candida auris have identified ERG genes involved in ergosterol biosynthesis as the major targets conferring rs-AMB. The results of this study also show that nonsynonymous mutations in ERG2 impair its function, abolish ergosterol from C. kefyr, and confer rs-AMB. Thus, rapid detection of rs-AMB among clinical isolates will help in proper management of invasive C. kefyr infections., Competing Interests: The authors declare no conflict of interest.

Background: Candida auris is an emerging, potentially multidrug-resistant pathogen that exhibits clade-specific resistance to fluconazole and also develops resistance to echinocandins and amphotericin B easily. This study analysed 49 C auris isolates for alterations in hotspot-1 and hotspot-2 of FKS1 for the detection of mutations conferring reduced susceptibility to echinocandins., Methods: C auris isolates (n = 49) obtained from 18 immunocompromised patients during June 2016-December 2018 were analysed. Antifungal susceptibility testing was performed by Etest and broth microdilution-based MICRONAUT-AM assay. Mutations in hotspot-1 and hotspot-2 regions of FKS1 were detected by PCR sequencing and fingerprinting of the isolates was done by short tandem repeat typing., Results: The patients had multiple comorbidities/risk factors for Candida spp. infection including cancer/leukaemia/lymphoma/myeloma (n = 16), arterial/central line (n = 17), urinary catheter (n = 17), mechanical ventilation (n = 14) and major surgery (n = 9) and received antifungal drugs as prophylaxis and/or empiric treatment. Seven patients developed C auris candidemia/breakthrough candidemia, nine patients had candiduria with/without candidemia and four patients developed surgical site/respiratory infection. Resistance to fluconazole and amphotericin B was detected in 44 and four isolates, respectively. Twelve C auris isolates from eight patients showed reduced susceptibility to echinocandins. Seven isolates contained hostspot-1 mutations and three isolates from a candidemia patient contained R1354H mutation in hotspot-2 of FKS1. Ten patients died, five were cured, two were lost to follow-up and treatment details for one patient were not available., Conclusions: Our findings describe development of a novel mutation in FKS1 conferring reduced susceptibility to echinocandins in one patient during treatment and unfavourable clinical outcome for many C auris-infected patients., (© 2021 Wiley-VCH GmbH.)

Objective: C. nivariensis is a rare Candida species which is phenotypically closely related to Candida glabrata and Candida bracarensis. The 3 species form the C. glabrata sensu lato complex. Here, we describe the first isolation and characterization of a C. nivariensis isolate cultured from the tracheal aspirate obtained from a young man in Kuwait., Materials and Methods: The yeast isolate was initially tested by VITEK 2 followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multiplex PCR. The identification was confirmed by sequencing of internal transcribed spacer (ITS) region of rDNA. Antifungal susceptibility testing was performed by Etest, and phylogenetic comparison with other international strains was carried out by using MEGA version 7 software., Results: The C. nivariensis isolate was misidentified by VITEK 2, but correctly identified by MALDI-TOF MS with updated software and multiplex PCR. The identity was confirmed by sequence comparisons of ITS region of rDNA. Antifungal susceptibility testing revealed high minimum inhibitory concentration (MIC) against fluconazole, but low MICs against amphotericin B and echinocandins. Phylogenetically, our isolate was closely related to Indian isolates., Conclusions: This report extends the geographic distribution of C. nivariensis to the Arabian Peninsula. MALDI-TOF MS with updated software and molecular tests are needed to correctly identify C. nivariensis. Since C. nivariensis may exhibit reduced susceptibility to antifungal agents, accurate identification and antifungal susceptibility testing are essential, particularly for isolates from sterile sites, for optimal patient management., (© 2020 The Author(s) Published by S. Karger AG, Basel.)

Objective: Candida kefyr causes invasive candidiasis in immunocompromised patients, particularly among those with oncohematological diseases. This study determined the prevalence of C. kefyr among yeast isolates collected during 2011-2018 in Kuwait. Antifungal susceptibility testing (AST) and genotypic heterogeneity among C. kefyr was also studied., Methods: Clinical C. kefyr isolates recovered from bloodstream and other specimens during 2011 to 2018 were retrospectively analyzed. All C. kefyr isolates were identified by CHROMagar Candida, Vitek2 and PCR amplification of rDNA. AST was performed by Etest. Molecular basis of resistance to fluconazole and echinocandins was studied by PCR-sequencing of ERG11 and FKS1, respectively. Genotypic heterogeneity was determined with microsatellite-/minisatellite-based primers and for 27 selected isolates by PCR-sequencing of IGS1 region of rDNA., Results: Among 8257 yeast strains, 69 C. kefyr (including four bloodstream) isolates were detected by phenotypic and molecular methods. Isolation from urine and respiratory samples from female and male patients was significantly different (P = 0.001). Four isolates showed reduced susceptibility to amphotericin B and one isolate to all (amphotericin B, fluconazole, voriconazole and caspofungin/micafungin) antifungals tested. Fluconazole-resistant isolate contained only synonymous mutations in ERG11. Echinocandin-resistant isolate contained wild-type hotspot-1 and hotspot-2 of FKS1. Fingerprinting with microsatellite-/minisatellite-based primers identified only three types. IGS1 sequencing identified seven haplotypes among 27 selected isolates., Conclusions: The overall prevalence of C. kefyr among clinical yeast isolates and among candidemia cases was recorded as 0.83% and 0.32%, respectively. The frequency of isolation of C. kefyr from bloodstream and other invasive samples was stable during the study period. The C. kefyr isolates grown from invasive (bloodstream, bronchoalveolar lavage, abdominal drain fluid, peritonial fluid and gastric fluid) samples and amphotericin B-resistant isolates were genotypically heterogeneous strains., Competing Interests: The authors have declared that no competing interests exist.

Changing trends in incidence and antifungal susceptibility patterns of six Candida species causing candidemia in Kuwait between 2006-2017 are reported. A total of 2075 isolates obtained from 1448 patients were analyzed. Identity of Candida species isolates was determined by phenotypic methods and confirmed by PCR amplification/PCR-sequencing of rDNA and/or MALDI-TOF MS. Antifungal susceptibility was determined by Etest. C. albicans accounted for 539 (37.22%) cases followed by C. parapsilosis (n = 502, 34.67%), C. tropicalis (n = 210, 14.5%), C. glabrata (n = 148, 10.22%), C. krusei (n = 27, 1.81%) and C. dubliniensis (n = 22, 1.5%). The comparative percent distribution of Candida species causing candidemia between 2006-2011 and 2012-2017 was as follows: C. albicans 41.8% and 33.1%, C. parapsilosis complex 32.01% and 37.04%, C. tropicalis 13.59% and 15.31%, and C. glabrata 8.77% and 11.51%, C. krusei 2.0% and 1.7%, and C. dubliniensis 1.75 and 1.3%, respectively. Three of 371 C. albicans isolates during 2006-2011 and five of 363 during 2012-2017 were resistant to fluconazole. Among C. parapsilosis isolates, one of 310 during 2006-2011 and 21 of 446 during 2012-2017 were resistant to this drug. Furthermore, at an epidemiologic cutoff value (ECV) of ≤0.5 μg/ml, 70.1% C. albicans isolates were wild-type for fluconazole during 2006-2011 as compared to 58.1% during 2012-2017. Likewise, at an ECV of ≤2 μg/ml, 98.0% of C. parapsilosis isolates were wild-type during 2006-2011 as compared to 93.4% during 2012-2017. Clonal spread of fluconazole-resistant C. parapsilosis in one major hospital was documented. An 8.8% shift in favor of non-albicans Candida species with concomitant increase in MICs between the two periods preludes emergence of fluconazole-resistant candidemia cases in Kuwait., Competing Interests: The authors have declared that no competing interests exist.

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.

Fluconazole resistance among clinical Candida parapsilosis isolates is an emerging problem in many countries, including Kuwait. Resistance to fluconazole is mediated by amino acid substitutions in ERG11 and/or by overexpression of efflux pumps MDR1 and CDR1. Clinical C. parapsilosis sensu stricto isolates (n = 442) were tested for susceptibility to fluconazole by Etest, Vitek II, and broth microdilution methods. ERG11 was analyzed from fluconazole-resistant, fluconazole-susceptible dose-dependent, and selected fluconazole-susceptible isolates. Of 442 C. parapsilosis isolates, 425, 2, and 15 were identified as susceptible, susceptible dose-dependent, and resistant to fluconazole, respectively. PCR sequencing of ERG11 identified Y132F mutation in 5 of 11 fluconazole-resistant isolates available for analysis. This mutation was absent in 46 fluconazole-susceptible and 2 fluconazole-susceptible dose-dependent isolates. A multiplex allele-specific PCR was developed for detection of Y132F mutation in ERG11, and results correlated perfectly with PCR sequencing data for ERG11 codon 132 for all isolates analyzed. Detection of resistance in 15 and reduced susceptibility in 2 among 442 C. parapsilosis isolates highlights emerging resistance to fluconazole in Kuwait. The Y132F mutation in ERG11 was found in 5 of 11 (45%) fluconazole-resistant isolates only. Detection of fluconazole resistance in C. parapsilosis will help in proper management of patients infected with this species.

Candida dubliniensis and Candida albicans are two closely related species. Although C. dubliniensis is less pathogenic, it has a higher propensity to develop resistance to fluconazole and some strains exhibit intrinsic resistance to 5-flucytosine (5-FC). All 5-FC-resistant isolates from Kuwait were previously shown to belong to one of seven internal transcribed spacer (ITS) region of rDNA-based haplotypes. This study performed fingerprinting of C. dubliniensis isolates by multilocus sequence typing (MLST) to determine population structure of 5-FC-resistant and -susceptible strains and compared the results with data from a global collection of isolates. Fifty-two C. dubliniensis isolates previously analyzed and 58 additional isolates mostly collected during 2010-2013 and characterized by phenotypic and molecular methods were used. ITS-based haplotypes were identified by haplotype-specific PCR and/or by PCR-DNA sequencing of rDNA. Population structure was determined by 8-loci-based MLST. E-test was used to determine susceptibility to 5-FC, fluconazole, voriconazole and amphotericin B. Five ITS haplotypes (ITSH) were detected among 110 C. dubliniensis isolates. The ITSH1 was most common (n = 80 isolates) followed by ITSH4 (n = 25 isolates). Two isolates each belonged to ITSH5 and ITSH8 while one isolate belonged to ITSH7. MLST identified 16 diploid sequence types (DSTs) including six new DSTs. DST11 (n = 52) and DST14 (n = 25) were dominant genotypes and were confined (together with DST21) to Middle-Eastern countries. Other DSTs (excluding some new DSTs) had a wider global distribution as they were identified from various other countries. Only ITSH4 isolates (n = 25) belonged to DST14, were resistant to 5-FC and contained S29L mutation in CdFCA1. ITSH5, ITSH7 and ITSH8 isolates belonged to different DSTs. Thus, clinical C. dubliniensis isolates in Kuwait exhibited limited genotypic heterogeneity and most isolates belonged to region-specific DSTs. All 5-FC-resistant C. dubliniensis isolates belonged to ITSH4 and MLST-based DST14 genotype. Placement of some isolates into additional ITS haplotypes is also supported by MLST data.

There is a paucity of information about genotypic heterogeneity among Candida dubliniensis isolates recovered from different geographic regions. This study explored genotypic heterogeneity among 103 C. dubliniensis strains obtained over a six-year period from clinical specimens in Kuwait. Genotype assignment was based on amplification with genotype-specific primers and sequencing of rDNA. Susceptibility to 5-flucytosine was determined by means of the Etest. DNA sequencing of cytosine deaminase was performed to determine the molecular basis of resistance to 5-flucytosine. DNA sequencing of rDNA identified seven different genotypes, i.e., 68 (66%) isolates were found to belong to genotype 1, 25 to genotype 4, six to genotype 5 and one each to genotypes 6-9. Strains of genotype 2 or genotype 3 were not detected. All isolates of genotype 4 but none of other genotypes were resistant to 5-flucytosine and the resistant strains all contained S29L mutation. Isolates of all other genotypes contained wild-type codon 29 in cytosine deaminase. A simple, PCR-RFLP-based method has been developed to facilitate rapid detection of S29L mutation in cytosine deaminase. A noteworthy observation of our study is the identification of five new genotypes of C. dubliniensis isolates, recovered from oral/respiratory specimens from patients of Middle Eastern origin. Furthermore, all 5-flucytosine resistant C. dubliniensis isolates in Kuwait belonged to genotype 4 only.

This study presents data on antifungal susceptibility of 114 Candida parapsilosis isolates recovered from clinical specimens in Kuwait. Candida parapsilosis isolates originating from blood (n = 66) and other clinical specimens (n = 48) were tested by Etest against amphotericin B (AP), caspofungin (CS), 5-flucytosine (FC), fluconazole (FL) and voriconazole (VO). The plates were incubated at 35 degrees C and readings for minimum inhibitory concentrations (MIC) were recorded after 24 and 48 h of incubation. The MIC ranges and MIC(90) read after 48 h were as follows: 0.064-1 and 0.5 microg ml(-1) for AP; 0.125-4 and 1.5 microg ml(-1) for CS; 0.047 to >256 and 1 microg ml(-1) for FL; 0.023 to >32 and 0.125 microg ml(-1) for FC and <0.002-1 and 0.047 microg ml(-1) for VO respectively. According to Clinical Laboratory Standards Institute criteria, all the isolates were susceptible to VO, and resistance against FC and FL was <2%. Eight (7%) isolates exhibited reduced susceptibility (MIC >1 microg ml(-1)) to CS including six isolates with MIC of >or=2 microg ml(-1) at 48 h reading. The antifungal resistance among bloodstream isolates of C. parapsilosis against AP, FL, FC and VO in Kuwait is rare. This is the first report on CS susceptibility of C. parapsilosis isolates from Arabian Gulf region.