Candida auris in various hospitals across Kuwait and their susceptibility and molecular basis of resistance to antifungal drugs.

BACKGROUND: Candida auris, a multidrug-resistant species, has the propensity of nosocomial transmission despite normal decontamination procedures. Here, we describe the isolation of C auris from patients in various hospitals in Kuwait during 2014-2018. Susceptibility to antifungal drugs and molecular basis of resistance to fluconazole, voriconazole and micafungin were also studied. METHODS: Candida auris (n = 314) obtained from 126 patients in eight hospitals were studied. All isolates were identified by PCR amplification and/or PCR-sequencing of ribosomal DNA (rDNA). Antifungal susceptibility was determined by Etest. Molecular basis of resistance to fluconazole and micafungin was studied by PCR-sequencing of ERG11 and FKS1 genes, respectively. FINDINGS: Bloodstream (n = 58), urine (n = 124), respiratory (n = 98) and other (n = 34) specimens yielded 314 C auris isolates. The proportion of bloodstream C auris among all yeast isolates was higher (42 of 307, 13.7%) in 2018 as compared to 2014-2017 (16 of 964, 1.7%) (P = .001). More bloodstream isolates (42 of 139) were cultured in 2018 than during 2014-2017 (16 of 175) (P = .001). Resistance to amphotericin B, fluconazole, voriconazole and micafungin was detected in 27.1%, 100%, 41.1% and 1.7% isolates, respectively. Fluconazole-resistant isolates contained either Y132F or K143R mutation in ERG11. Isolates with K143R mutation were additionally resistant to voriconazole. Micafungin-resistant isolates contained S639F mutation in hot spot 1 of FKS1. CONCLUSIONS: Our study highlights spreading of C auris in major hospitals across Kuwait and its increasing role as a bloodstream pathogen in 2018. Cross-resistance to voriconazole was also seen in isolates with K143R mutation in ERG11, while micafungin-resistant isolates harboured S639F mutation in hot spot 1 of FKS1.

ERG6 and ERG2 Are Major Targets Conferring Reduced Susceptibility to Amphotericin B in Clinical Candida glabrata Isolates in Kuwait.

Candida glabrata is intrinsically less susceptible to azoles, and resistance to echinocandins and reduced susceptibility (RS) to amphotericin B (AMB) have also been detected. The molecular mechanisms of RS to AMB were investigated in C. glabrata strains in Kuwait by sequence analyses of genes involved in ergosterol biosynthesis. A total of 1,646 C. glabrata isolates were tested by Etest, and results for 12 selected isolates were confirmed by reference broth microdilution. PCR sequencing of three genes (ERG2, ERG6, and ERG11) was performed for all isolates with RS to AMB (RS-AMB isolates) and 5 selected wild-type C. glabrata isolates by using gene-specific primers. The total cell sterol content was analyzed by gas chromatography-mass spectrometry. The phylogenetic relationship among the isolates was investigated by multilocus sequence typing. Wild-type isolates contained only synonymous mutations in ERG2, ERG6, or ERG11, and the total sterol content was similar to that of the reference strains. A nonsynonymous ERG6 mutation (AGA48AAA, R48K) was found in both RS-AMB and wild-type isolates. Four RS-AMB isolates contained novel nonsense mutations at Trp286, Tyr192, and Leu341, and 2 isolates contained a nonsynonymous mutation in ERG6 (V126F or C198F); and the sterol content of these isolates was consistent with ERG6 deficiency. Two other RS-AMB isolates contained a novel nonsynonymous ERG2 mutation (G119S or G122S), and their sterol content was consistent with ERG2 deficiency. Of 8 RS-AMB isolates, 1 fluconazole-resistant isolate also contained nonsynonymous Y141H plus L381M mutations, while 7 isolates contained only synonymous mutations in ERG11 All isolates with ERG6, ERG2, and ERG11 mutations were genotypically distinct strains. Our data show that ERG6 and ERG2 are major targets conferring RS-AMB in clinical C. glabrata isolates.

Cyberlindnera fabianii fungaemia outbreak in preterm neonates in Kuwait and literature review.

BACKGROUND: Cyberlindnera fabianii has rarely been reported as a human pathogen. Here, we describe an outbreak of C. fabianii fungaemia involving 10 preterm neonates during a seven-month period in Kuwait and review the published reports. METHODS: Blood cultures were processed, and yeast isolates were initially identified by ID 32 C and/or VITEK 2. Molecular identification was done by PCR sequencing of internally transcribed spacer (ITS) region and D1/D2 domains of rDNA. Fingerprinting was performed with microsatellite-based and minisatellite-based primers to examine genetic relatedness among the isolates. Antifungal susceptibility testing of the isolates was done by Etest. FINDINGS: All infected neonates were preterm, received prior antibiotics and had an intravascular catheter in place. All bloodstream isolates were initially identified as Candida utilis by ID 32 C and/or VITEK 2 and showed reduced susceptibility to triazoles. PCR sequencing of rDNA identified all isolates as Cyberlindnera fabianii. Fingerprinting studies yielded identical patterns indicating clonality. One neonate died before treatment, one died during treatment, and eight neonates survived treatment with amphotericin B with/without fluconazole or caspofungin. Source of infection remained unknown despite surveillance cultures. CONCLUSION: The outbreak highlights emergence of C. fabianii as a neonatal pathogen and reinforces importance of molecular methods in its accurate identification.

Demonstration of Adventitious Sporulation in Fusarium Petroliphilum Onychomycosis.

Fusarium petroliphilum is a recently described species within the Fusarium solani species complex. Some strains of Fusarium species are capable of forming yeast-like structures in tissue as well as in culture through a process known as "adventitious sporulation." Here, we describe the formation of these yeast-like reproductive structures in infected nail tissue obtained from a case of onychomycosis. These structures were also observed in culture grown on RPMI 1640 agar supplemented with 2% glucose. The isolate was resistant to azoles and echinocandins. To the best of our knowledge, this is the first report describing adventitious sporulation in F. petroliphilum and its etiologic role in onychomycosis.

Allergic Fungal Sinusitis Caused by Exserohilum rostratum and Literature Review.

A case of allergic fungal sinusitis (AFS) caused by Exserohilum rostratum, proven by culture and histopathology of the biopsy material, is described. The identity of the isolate was confirmed by sequencing of ITS region of rDNA. To the best of our knowledge, this is the first report of allergic E. rostratum sinusitis from Kuwait and Arabian Peninsula. Ten previously described cases of AFS have been reviewed. The report highlights the emerging importance of E. rostratum as a human pathogen in this region and role of molecular methods in its accurate identification.

In vitro Post-Antifungal Effect of Posaconazole and Its Impact on Adhesion-Related Traits and Hemolysin Production of Oral Candida dubliniensis Isolates.

OBJECTIVE: Candidal adherence to denture acrylic surfaces (DAS) and oral buccal epithelial cells (BEC), formation of candidal germ tubes (GT), candidal cell surface hydrophobicity (CSH), and hemolysin production are important pathogenic traits of Candida. The antifungal drug-induced post-antifungal effect (PAFE) also impacts the virulence of Candida. Candida dubliniensis isolates are associated with the causation of oral candidiasis which could be managed with posaconazole. Thus far there is no evidence on posaconazole-induced PAFE and its impact on adhesion-related attributes and production of hemolysin by C. dubliniensis isolates. Hence, the PAFE, adhesion to DAS and BEC, formation of GT, CSH, and hemolysin production of 20 oral C. dubliniensis isolates after brief exposure to posaconazole was ascertained. MATERIALS AND METHODS: The PAFE, adherence to DAS and BEC, formation of GT, candidal CSH, and hemolysin production were investigated by hitherto described in vitro assays. RESULTS: The mean PAFE (h) induced by posaconazole on C. dubliniensis isolates was 1.66. Exposure to posaconazole suppressed the ability of C. dubliniensis to adhere to DAS, BEC, formation of candidal GT, candidal CSH and to produce hemolysin by a reduction of 44, 33, 34, 36, and 15% (p < 0.005 to p < 0.001), respectively. CONCLUSION: Exposure of C. dubliniensis isolates to posaconazole for a brief period induced an antimycotic impact by subduing its growth in addition to suppressing pathogenic adherence-associated attributes, as well as production of hemolysin.

Development of Echinocandin Resistance in Candida tropicalis following Short-Term Exposure to Caspofungin for Empiric Therapy.

Isolation of two echinocandin-resistant Candida tropicalis strains from endotracheal secretions of a patient following short-term exposure to caspofungin is described. Both strains exhibited resistance to echinocandins by Etest and reference broth microdilution, showing a homozygous S645P mutation within the hot spot 1 (HS-1) region of FKS1 and belonging to a unique multilocus sequence type. Other C. tropicalis isolates collected from patients in the same intensive care unit within a 60-day period were susceptible to echinocandins and contained wild-type FKS1 sequences.

Invasive Candida auris infections in Kuwait hospitals: epidemiology, antifungal treatment and outcome.

PURPOSE: Candida auris is a recently recognized yeast pathogen, which has attracted worldwide attention due to its multidrug-resistant nature and associated high mortality rates. Its persistence in hospital environment and propensity of nosocomial transmission underscores the need of continuous monitoring to prevent outbreaks. Since the first case of C. auris candidemia in May, 2014, we have identified 17 additional invasive cases, which are described here. METHODS: Identity of 17 isolates originating from proven or possible cases of invasive C. auris infection and identified as Candida haemulonii by Vitek 2 yeast identification system was confirmed by PCR-sequencing of rDNA. Information about risk factors, treatment and outcomes were retrospectively retrieved from case files. Antifungal susceptibility testing was performed by Etest. RESULTS: Thirteen cases of candidemia and 4 cases of other invasive infections were detected in 6 hospitals across Kuwait. Major risk factors included adult patients with cancer, diabetes, gastrointestinal/liver diseases and extended (> 25 days) hospital stay. All isolates were resistant to fluconazole. Additionally, 5 and 4 isolates were also resistant to voriconazole and amphotericin B, respectively. Despite antifungal treatment, 9 of 15 patients died. Most patients (n = 12) were hospitalized in 2 hospitals that are in close proximity, whereas 5 other patients were from 3 hospitals that are situated > 10 km apart. CONCLUSIONS: Occurrence of successive cases of invasive C. auris infections with resulting mortality in nine patients suggests persistence of this multidrug-resistant yeast in major hospitals in Kuwait. Early detection by continuous surveillance and enforcement of infection control measures are recommended.

Rapid and Accurate Identification of Candida albicans and Candida dubliniensis by Real-Time PCR and Melting Curve Analysis.

OBJECTIVE: Candida albicans and Candida dubliniensis are germ tube-positive pathogenic yeast species. Accurate identification of these two species is warranted since C. albicans is a highly pathogenic species while C. dubliniensis exhibits increased adherence to buccal epithelial cells, reduced susceptibility to azoles and resistance to flucytosine. We have developed a duplex real-time PCR assay for rapid detection and differentiation between clinical C. albicans and C. dubliniensis isolates. MATERIALS AND METHODS: A duplex real-time PCR assay was developed by using two species-specific primer pairs and SYBR Green dye to differentiate C. albicans and C. dubliniensis isolates via melting curve analysis of real-time PCR amplicons. Amplification products were also analyzed by agarose gel electrophoresis to confirm real-time PCR results. RESULTS: Melting temperatures (Tm) for reference strains of C. albicans and C. dubliniensis were 86.55 and 82.75°C, respectively. No amplicon was obtained with DNA from reference strains of 8 other common Candida spp. When real-time PCR was applied on 226 clinical isolates previously identified by the Vitek 2 system and/or PCR sequencing of rDNA, Tm values for C. albicans (n = 113) and C. dubliniensis (n = 98) were 86.68 ± 0.529 and 82.616 ± 0.535°C, respectively. The results were confirmed by agarose gel electrophoresis. No amplicon was obtained from 15 isolates belonging to 9 other Candida spp. CONCLUSIONS: The real-time PCR assay described here does not require prior identification of clinical yeast isolates as C. albicans/C. dubliniensis by germ tube formation and accurately reports results within 2 h. Detection of amplicons by agarose gel electrophoresis is also suitable for resource-poor settings devoid of real-time PCR facilities.

Impact of Brief Exposure to Drugs with Antifungal Properties on the Susceptibility of Oral Candida dubliniensis Isolates to Lysozyme and Lactoferrin.

OBJECTIVE: Lysozyme and lactoferrin have anti-candidal activity. Candida dubliniensis is associated with oral candidiasis. Candida infections are managed with nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine. Candida species undergo a brief exposure to therapeutic agents in the mouth. There is no data on the influence of limited exposure to antimycotics on the sensitivity of C. dubliniensis to lactoferrin and lysozyme. Hence, this study observed the changes in the sensitivity of C. dubliniensis to anti-candidal action of lactoferrin and lysozyme after transitory exposure to sub-lethal concentrations of antifungals. MATERIALS AND METHODS: After determination of the minimum inhibitory concentration (MIC), 20 C. dubliniensis isolates were exposed to twice the concentration of MIC of nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine for 1 h. Drugs were removed by dilution and thereafter the susceptibility of these isolates to lysozyme and lactoferrin was determined by colony-forming unit quantification assay. RESULTS: Exposure of C. dubliniensis to nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine resulted in an increase in susceptibility to lysozyme by 9.45, 30.82, 30.04, 50.64, 55.60, and 50.18%, respectively (p < 0.05 to p < 0.001). Exposure of C. dubliniensis to nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine resulted in an increase in susceptibility to lactoferrin by 13.54, 16.43, 17.58, 19.60, 21.32, and 18.73, respectively (p < 0.05 to p < 0.001). CONCLUSION: Brief exposure to nystatin, amphotericin B, caspofungin, ketoconazole, fluconazole, and chlorhexidine enhances the antifungal effect of lysozyme and lactoferrin on C. dubliniensis isolates in vitro.

Candida fermentati as a cause of persistent fungemia in a preterm neonate successfully treated by combination therapy with amphotericin B and caspofungin.

A case of persistent candidemia in a preterm neonate caused by Candida fermentati, identified by sequencing of the internally transcribed spacer region of ribosomal DNA (rDNA), is described. The neonate was treated for 30 days by combination therapy with amphotericin B (AmBisome) and caspofungin with a successful outcome, and no drug-related side effects were observed.

Concomitant occurrence of itraconazole-resistant and -susceptible strains of Aspergillus fumigatus in routine cultures.

OBJECTIVES: Resistance to triazoles in Aspergillus fumigatus has emerged in several countries and is usually mediated by mutations in the cyp51A gene. We determined the presence of both itraconazole-susceptible and -resistant A. fumigatus in routine cultures isolated from environmental and clinical samples. METHODS: A total of 50 environmental and 16 clinical A. fumigatus isolates obtained from single colonies were tested for itraconazole susceptibility by Etest. Serial dilution and plating for selected A. fumigatus cultures were performed to separate triazole-susceptible and -resistant phenotypes. Antifungal drug susceptibility of subcultures to itraconazole, posaconazole and voriconazole was determined by the broth microdilution method. Itraconazole resistance was determined by the presence of tandem repeats in the promoter region and other resistance-conferring mutations by PCR and/or direct DNA sequencing of cyp51A. Genotyping was performed with a panel of nine microsatellite loci. RESULTS: PCR amplification of the promoter region identified 1 of 50 environmental and 2 of 16 clinical A. fumigatus isolates as mixed (itraconazole-resistant and -susceptible) cultures, while the remaining isolates yielded patterns that were consistent with their data for susceptibility to itraconazole. Purified subcultures yielded distinct susceptibility profiles, concomitant with genetic determinants of triazole susceptibility/resistance in cyp51A and different microsatellite patterns. CONCLUSIONS: Concomitant presence of triazole-susceptible and -resistant strains in single colonies of routine A. fumigatus cultures, obtained from environmental and clinical samples, has been conclusively demonstrated.

Aerial prevalence of Aspergillus calidoustus isolates in and around a tertiary care hospital in Kuwait and assessment of their pathogenicity.

Seven Aspergillus calidoustus isolates from 486 Aspergillus spp. isolates (1.4% overall prevalence) from outdoor/indoor air samples and one isolate from the bronchoalveolar lavage fluid of a patient with pneumonia were obtained. These 8 isolates exhibited reduced susceptibility to triazoles. Preliminary pathogenicity data from BALB/c mice suggest that A. calidoustus can persist in tissues for long periods without causing mortality. Further studies using graded doses of inoculum and immunosuppression models are warranted to gain an understanding of the factors associated with its pathogenicity and virulence.

Simple, low-cost molecular assays for TR34/L98H mutations in the cyp51A gene for rapid detection of triazole-resistant Aspergillus fumigatus isolates.

Simple, low-cost PCR/PCR-restriction fragment length polymorphism (RFLP) assays targeting cyp51A promoter and codon 98 regions were developed for the detection of triazole-resistant Aspergillus fumigatus strains carrying TR34/L98H mutations. The assays were evaluated using 40 itraconazole-susceptible isolates and 35 itraconazole-resistant isolates. The prevalence of TR34/L98H mutations in clinical/environmental A. fumigatus isolates may now be determined easily from resource-poor settings.

Isolation of cholesterol-dependent, multidrug-resistant Candida glabrata strains from blood cultures of a candidemia patient in Kuwait.

BACKGROUND: Candida glabrata has emerged as an important human pathogen associated with systemic and mucosal infections. Here, we describe isolation of two cholesterol-dependent Candida glabrata strains from a candidemia patient which failed to grow on the media devoid of a cholesterol source. METHODS: Both the isolates were recovered from BACTEC Plus Aerobic/F blood culture bottles of a candidemic patient. Since these isolates failed to grow on Sabouraud dextrose agar, Mueller-Hinton agar and RPMI 1640 agar media, their definitive identification required PCR sequencing of the internally transcribed spacer (ITS)1 and ITS2 regions of rDNA and the D1/D2 region sequences within 26S rRNA gene. The cholesterol auxotrophy was determined by their ability to grow on media containing a cholesterol source. The minimum inhibitory concentrations (MICs) to antifungal agents were determined by Etest. RESULTS: The identity of the isolates was confirmed by sequencing of the ITS1 and ITS2 regions of rDNA and the D1/D2 region sequences within 26S rRNA gene and also by matrix-assisted laser desorption and ionization-time-of-flight mass spectrometry with 99.9% confidence value. Both the isolates showed good growth only when media were supplemented with cholesterol, oxbile or blood. Additionally, these isolates were resistant to amphotericin B (MIC ≥32 µg/ml), fluconazole (MIC ≥256 µg/ml), voriconazole (MIC ≥32 µg/ml), itraconazole (MIC ≥32 µg/ml), and posaconazole (MIC ≥32 µg/ml), but susceptible to caspofungin (MIC range 0.064 to 0.19 µg/ml). CONCLUSION: This appears to be the first report on isolation of cholesterol-dependent strains of C. glabrata from a candidemia patient exhibiting resistance to azoles and amphotericin B. Further, the report demonstrates that induction of cholesterol/sterol auxotrophy is associated with resistance to antifungal drugs targeting ergosterol biosynthesis. These observations may have therapeutic implications for the treatment of infections caused by such C. glabrata strains.

Occurrence of triazole-resistant Aspergillus fumigatus with TR34/L98H mutations in outdoor and hospital environment in Kuwait.

BACKGROUND: Invasive aspergillosis due to triazole-resistant Aspergillus fumigatus, a common airborne fungal pathogen, has emerged in some European and Asian countries, likely due to exposure to azole fungicides in the environment. This study determined the occurrence of triazole-resistant A. fumigatus in outdoor and hospital environment in Kuwait, a semi-arid desert country in Arabian Peninsula. METHODS: Outdoor/indoor air, water and cotton swab samples were cultured on malt extract agar and A. fumigatus colonies were identified by phenotypic and molecular methods. Drug susceptibility of A. fumigatus isolates to itraconazole, posaconazole and voriconazole was carried out by an Etest and a broth microdilution method. Resistance mechanisms involving cyp51A mutations were probed by mixed-format real-time (MF-rt)-PCR assays. Triazole-resistant isolates were typed by nine-locus microsatellite analysis. A multiplex allele-specific (MAS)-PCR assay was developed for detection of L98H mutation in cyp51A. RESULTS: Of 115 A. fumigatus isolates obtained from 362 environmental samples from across Kuwait, 8 isolates were resistant to itraconazole, posaconazole and voriconazole. All itraconazole-resistant isolates contained a 34-bp tandem repeat (TR34) in the promoter region and a L98H mutation at codon 98 (TR34/L98H) in cyp51A. These mutations were absent in all itraconazole-susceptible isolates. MAS-PCR accurately detected L98H mutation in all triazole-resistant isolates. Three microsatellite patterns were observed among resistant isolates with one pattern clustering with Indian clinical and environmental isolates. CONCLUSIONS: Triazole-resistant A. fumigatus with TR34/L98H mutations in cyp51A is prevalent in Kuwait. Although triazole-resistant A. fumigatus has not yet been isolated from clinical specimens, its presence in the environment suggests that the possibility of susceptible individuals getting infected with such strains exists and may pose therapeutic challenges in its management.

Spectrum and management of rare Candida/yeast infections in Kuwait in the Middle East.

Invasive fungal infections (IFIs) are associated with high mortality rates and mostly affect patients with compromised immunity. The incidence of IFIs is increasing worldwide with the expanding population of susceptible patients. Candida and other yeast infections represent a major component of IFIs. Rare Candida/yeast infections have also increased in recent years and pose considerable diagnostic and management challenges as they are not easily recognized by routine phenotypic characteristic-based diagnostic methods and/or by the automated yeast identification systems. Rare Candida/yeasts also exhibit reduced susceptibility to antifungal drugs making proper management of invasive infections challenging. Here, we review the diagnosis and management of 60 cases of rare Candida/yeast IFIs described so far in Kuwait, an Arabian Gulf country in the Middle East. Interestingly, majority (34 of 60, 56.7%) of these rare Candida/yeast invasive infections occurred among neonates or premature, very-low-birth-weight neonates, usually following prior bacteremia episodes. The clinical details, treatment given, and outcome were available for 28 of 34 neonates. The crude mortality rate among these neonates was 32.2% as 19 of 28 (67.8%) survived the infection and were discharged in healthy condition, likely due to accurate diagnosis and frequent use of combination therapy. Physicians treating patients with extended stay under intensive care, on mechanical ventilation, receiving broad spectrum antibiotics and with gastrointestinal surgery/complications should proactively investigate IFIs. Timely diagnosis and early antifungal treatment are essential to decrease mortality. Understanding the epidemiology and spectrum of rare Candida/yeast invasive infections in different geographical regions, their susceptibility profiles and management will help to devise novel diagnostic and treatment approaches and formulate guidelines for improved patient outcome.

Molecular identification and antifungal susceptibility profile of Aspergillus flavus isolates recovered from clinical specimens in Kuwait.

BACKGROUND: Within the genus Aspergillus, A. flavus is the second most important species of clinical significance. It is predominantly associated with infections involving sinuses, eye and skin, mostly in geographic regions with hot and arid climate, including the Middle East. Recent reports on emergence of resistance to triazoles among Aspergillus spp. is a cause of concern for treatment of patients with invasive aspergillosis. In this study we present data on genetic characterization and antifungal susceptibility profile of clinical and environmental isolates of A. flavus. METHODS: Ninety-nine Aspergillus section Flavi isolates, originating from clinical (n=92) and environmental (n=7) sources, initially identified by morphological characteristics, were analyzed by partial sequencing of ß-tubulin and calmodulin gene fragments and their susceptibilities to six antifungal agents was determined by Etest on RPMI1640 and Muller-Hinton agar media. Etest minimum inhibitory concentrations (MICs) of amphotericin B and voriconazole were also compared with zone of inhibition diameters obtained by disc diffusion test on RPMI agar medium. RESULTS: The identity of all clinical and environmental isolates was confirmed as A. flavus species by combined analysis of ß-tubulin and calmodulin genes. The mean MIC90 (µg/ml) values on RPMI medium for amphotericin B, voriconazole, posaconazole, anidulafungin, micafungin and caspofungin were 3, 0.25, 0.25, 0.002, 0.002 and 0.032, respectively. No environmental isolate exhibited MIC value of >2 µg/ml for amphotericin B. For clinical isolates, the zone of inhibition diameters for amphotericin B and voriconazole ranged from 7-16 mm and 24-34 mm, respectively. Linear regression analysis between Etest MIC values and disk diffusion diameters revealed a significant inverse correlation with amphotericin B (p <0.001) and voriconazole (p<0.003). CONCLUSIONS: The ß-tubulin and calmodulin gene sequences confirmed that all 92 clinical isolates identified phenotypically belonged to A. flavus taxon, thus suggesting that the other species within Aspergillus section Flavi are of little clinical significance. Triazoles and echinocandins showed very good in vitro activity against the A. flavus, however, 10% clinical isolates showed MICs of >2 µg/ml for amphotericin B.

Coniochaeta polymorpha, a new species from endotracheal aspirate of a preterm neonate, and transfer of Lecythophora species to Coniochaeta.

A new species of Coniochaeta from endotracheal secretion of a preterm neonate, Coniochaeta polymorpha, is described. This anamorphic species is characterized by development of dark brown colonies after 1 week of incubation on culture medium, formation of abundant yeast-like cells and sclerotium-like structures producing discrete, brown, nearly globose phialidic conidiogenous cells and absence of chlamydospores. A combined sequence dataset of the ITS region, partial LSU rDNA, actin and ß-tubulin genes sufficiently resolved the unique phylogenetic status of this species. In response to recent changes in the nomenclature for pleomorphic fungi, we transfer the Lecythophora species to Coniochaeta, and propose the following new combinations: Coniochaeta canina, Coniochaeta cateniformis, Coniochaeta decumbens, Coniochaeta fasciculata, Coniochaeta hoffmannii, Coniochaeta lignicola, Coniochaeta luteorubra, Coniochaeta luteoviridis and Coniochaeta mutabilis.

Molecular fingerprinting by multi-locus sequence typing identifies microevolution and nosocomial transmission of Candida glabrata in Kuwait.

Backgrounds: Candida glabrata is a frequently isolated non-albicans Candida species and invasive C. glabrata infections in older patients are associated with high mortality rates. Opportunistic Candida infections in critically ill patients may be either endogenous or nosocomial in origin and this distinction is critical for effective intervention strategies. This study performed multi-locus sequence typing (MLST) to study genotypic relatedness among clinical C. glabrata isolates in Kuwait. Methods: Candida glabrata isolates (n = 91) cultured from 91 patients were analyzed by MLST. Repeat isolates (n = 16) from 9 patients were also used. Antifungal susceptibility testing for fluconazole, voriconazole, caspofungin and amphotericin B (AMB) was determined by Etest. Genetic relatedness was determined by constructing phylogenetic tree and minimum spanning tree by using BioNumerics software. Results: Resistance to fluconazole, voriconazole and AMB was detected in 7, 2 and 10 C. glabrata isolates, respectively. MLST identified 28 sequence types (STs), including 12 new STs. ST46 (n = 33), ST3 (n = 8), ST7 (n = 6) and ST55 (n = 6) were prevalent in ≥4 hospitals. Repeat isolates obtained from same or different site yielded identical ST. No association of ST46 with source of isolation or resistance to antifungals was apparent. Microevolution and cross-transmission of infection was indicated in two hospitals that yielded majority (57 of 91, 67%) of C. glabrata. Conclusion: Our data suggest that C. glabrata undergoes microevolution in hospital environment and can be nosocomially transmitted to other susceptible patients. Thus, proper infection control practices during routine procedures on C. glabrata-infected patients may prevent transmission of this pathogen to other hospitalized patients.