The emerging, often multidrug-resistant Candida auris is increasingly being associated with outbreaks in healthcare facilities. Here we describe the molecular epidemiology of a C. auris outbreak during 18 months, which started in 2018 in the high dependency unit (HDU) of a secondary-care hospital in Kuwait. Demographic and clinical data for candidemia and colonized patients were prospectively recorded. Clinical and environmental isolates were subjected to phenotypic and molecular identification; antifungal susceptibility testing by broth microdilution method; PCR-sequencing of ERG11 and FKS1 for resistance mechanisms to triazoles and echinocandins, respectively; and molecular fingerprinting by short tandem repeat (STR) analyses. Seventy-one (17 candidemic and 54 colonized) patients including 26 with candiduria and seven environmental samples yielded C. auris. All isolates were identified as C. auris by Vitek2, MALDI-TOF MS, PCR amplification and/or PCR-sequencing of rDNA. Twelve candidemia and 26 colonized patients were admitted or exposed to HDU. Following outbreak recognition, an intensive screening program was instituted for new patients. Despite treatment of all candidemia and 36 colonized patients, 9 of 17 candidemia and 27 of 54 colonized patients died with an overall crude mortality rate of ~50%. Nearly all isolates were resistant to fluconazole and contained the Y132F mutation in ERG11 except one patient’s isolates, which were also distinct by STR typing. Only urine isolates from two patients developed echinocandin resistance with concomitant FKS1 mutations. The transmission of C. auris in this outbreak was linked to infected/colonized patients and the hospital environment. However, despite continuous surveillance and enforcement of infection control measures, sporadic new cases continued to occur, challenging the containment efforts.

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.

Candida auris is an emerging yeast pathogen that has recently caused major outbreaks in healthcare facilities worldwide. Clinical C. auris isolates are usually resistant to fluconazole and readily develop resistance to echinocandins and amphotericin B (AMB) during treatment. We describe here an interesting case of C. auris infection in an immunocompromised patient who had previously received AMB and caspofungin treatment. Subsequently, C. auris was isolated from tracheal (tracheostomy) secretions and twice from urine and all three isolates were susceptible to AMB and micafungin. The patient received a combination therapy with AMB and caspofungin. Although the C. auris was cleared from the urine, the patient subsequently developed breakthrough candidemia and the bloodstream isolate exhibited a reduced susceptibility to micafungin and also showed the presence of a novel (S639T) mutation in hotspot-1 of FKS1. Interestingly, C. auris from the tracheal (tracheostomy) secretions recovered one and four days later exhibited a reduced susceptibility to micafungin and S639Y and S639T mutations in hotspot-1 of FKS1, respectively. Although the treatment was changed to voriconazole, the patient expired. Our case highlights a novel FKS1 mutation and the problems clinicians are facing to treat invasive C. auris infections due to inherent or developing resistance to multiple antifungal drugs and limited antifungal armamentarium.

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.

Occurrence of Candida nivariensis and Candida bracarensis, two species phenotypically similar to Candida glabrata sensu stricto, in human clinical samples from different geographical settings remains unknown. This study developed a low-cost multiplex PCR (mPCR) and three species-specific singleplex PCR assays. Reference strains of common Candida species were used during development and the performance of mPCR and singleplex PCR assays was evaluated with 440 clinical C. glabrata sensu lato isolates. The internal transcribed spacer (ITS) region of rDNA was also sequenced from 85 selected isolates and rDNA sequence variations were used for determining genetic relatedness among the isolates by using MEGA X software. Species-specific amplicons for C. glabrata (~360 bp), C. nivariensis (~250 bp) and C. bracarensis (~180 bp) were obtained in mPCR while no amplicon was obtained from other Candida species. The three singleplex PCR assays also yielded expected results with reference strains of Candida species. The mPCR amplified ~360 bp amplicon from all 440 C. glabrata sensu lato isolates thus identifying all clinical isolates in Kuwait as C. glabrata sensu stricto. The results of mPCR were confirmed for all 440 isolates as they yielded an amplicon only in C. glabrata sensu stricto-specific singleplex PCR assay. The rDNA sequence data identified 28 ITS haplotypes among 85 isolates with 18 isolates belonging to unique haplotypes and 67 isolates belonging to 10 cluster haplotypes. In conclusion, we have developed a simple, low-cost mPCR assay for rapid differentiation of C. glabrata sensu stricto from C. nivariensis and C. bracarensis. Our data obtained from a large collection of clinical C. glabrata sensu lato isolates show that C. nivariensis and C. bracarensis are rare pathogens in Kuwait. Considerable genetic diversity among C. glabrata sensu stricto isolates was also indicated by rDNA sequence analyses.