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.

Purpose of Review Candida auris , a recently recognized yeast pathogen, has become a major public health threat due to the problems associated with its accurate identification, intrinsic and acquired resistance to antifungal drugs, and its potential to easily contaminate the environment causing clonal outbreaks in healthcare facilities. These outbreaks are associated with high mortality rates particularly among older patients with multiple comorbidities under intensive care settings. The purpose of this review is to highlight strategies that are being adapted to prevent transmission of C. auris in healthcare settings. Recent Findings Colonized patients shed C. auris into their environment which contaminates surrounding equipment. It resists elimination even by robust decontamination procedures and is easily transmitted to new patients during close contact resulting in outbreaks. Efforts are being made to rapidly identify C. auris -infected/ C. auris -colonized patients, to determine its susceptibility to antifungals, and to perform effective cleaning and decontamination of the environment and isolation of colonized patients to prevent further transmission. Summary Rapid and accurate identification of hospitalized patients infected/colonized with C. auris , rapid detection of its susceptibility patterns, and appropriate use of infection control measures can help to contain the spread of this highly pathogenic yeast in healthcare settings and prevent/control outbreaks.

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.

The Candida species cause a majority of invasive fungal infections. In this article, we describe the nationwide epidemiology of candidemia in Kuwait in 2018. Yeast bloodstream isolates submitted from all major hospitals and identified by phenotypic MALDI-TOF MS and/or by molecular methods were studied. Susceptibility testing was performed by Etest. Out of 313 bloodstream yeasts, 239 Candida spp. isolates (excluding duplicate isolates) were obtained during 234 candidemic episodes among 223 patients. Mixed-species candidemia and re-infection occurred in 5 and 11 patients, respectively. C. albicans (n = 74), C. parapsilosis (n = 54), C. tropicalis (n = 35), C. auris (n = 33), C. glabrata (n = 32), other Candida spp. (n = 11), and other yeasts (n = 9) caused fungemia. Nearly 50% of patients were in intensive care units. Candida spp. isolates (except C. glabrata) were susceptible to caspofungin and 27% of C. auris were amphotericin B-resistant. Resistance to fluconazole was 100% in C. auris, 17% in C. parapsilosis, 12% in C. glabrata, and 1% in C. albicans. Mortality was 47% for other Candida/yeast infections. Nationwide candidemia incidence in 2018 was 5.29 cases/100,000 inhabitants. Changes in species spectrum, increasing fluconazole resistance in C. parapsilosis, and the emergence of C. auris as a major pathogen in Kuwait are noteworthy findings. The data could be of help in informing decisions regarding planning, in the allocation of resources, and in antimicrobial stewardship.

Introduction. Papiliotrema laurentii, formerly Cryptococcus laurentii, is typically isolated from environmental sources, but also occasionally from clinical specimens. Other close relatives may be misidentified as P. laurentii by phenotypic methods. P. laurentii usually lacks melanin; however, melanin-forming strains have also been isolated. Hypothesis/Gap Statement. Although melanin production by encapsulated budding yeasts is considered a major virulence factor, the comparative pathogenicity of melanin-forming and non-melanized environmental strains of P. laurentii has rarely been studied. Aim. We performed phenotypic and molecular identification and determined the genotypic heterogeneity among P. laurentii isolates. We also studied the pathogenicity of melanin-forming and non-melanized strains in normal and immunosuppressed mice. Methodology. Eleven environmental isolates were tested for their identity by Vitek2 and/or ID32C systems, and by PCR-sequencing of the internal transcribed spacer (ITS) region and D1/D2 domains of ribosomal DNA (rDNA). Genotypic heterogeneity was studied by sequence comparisons. The pathogenicity of melanized and non-melanized P. laurentii strains was studied in intravenously infected normal and immunosuppressed BALB/c mice. Results. Phenotypic methods identified seven of the environmental isolates, while PCR-sequencing of the ITS region and D1/D2 domains of rDNA detected two and five isolates, respectively, as P. laurentii. Sequence comparisons demonstrated genotypic heterogeneity among P. laurentii. The remaining four environmental isolates yielded expected results. None of the normal mice infected with 105 cells of melanized/non-melanized P. laurentii strains died. Infection of immunosuppressed mice with 107 cells caused higher mortality with non-melanized P. laurentii, while viable counts in brain/lung tissue were higher in mice infected with a melanized strain and were detectable for up to 14 days. Conclusion. Phenotypic methods lacked specificity, but PCR-sequencing of D1/D2 domains correctly identified P. laurentii and sequence comparisons demonstrated the genotypic heterogeneity of the isolates. Both melanized and non-melanized strains at a higher dose caused mortality in immunosuppressed mice and persisted in brain/lung tissue up to 14 days post-infection.