Identification of Candida dubliniensis in a Prospective Study of Patients in the United States

Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other yeast species are often identified in human immunodeficiency virus (HIV)-seropositive patients. Candida dubliniensis phenotypically resembles C. albicans in many respects, yet it can be identified and differentiated as a unique Candida species by its phenotypic and genetic profiles. The purpose of the present study was to prospectively test for the presence of C. dubliniensis among clinical isolates and to determine the clinical and demographic characteristics of patients harboring C. dubliniensis. Over a 90-day period, isolates from 724 patients that were presumptively identified as C. albicans were screened for C. dubliniensis by use of tests for germ tube and chlamydospore production, by detection of an inability to grow at 45°C, by colony color on CHROMagar Candida medium, and by the results of a sugar assimilation test with the API 20C AUX yeast identification system. Among 699 isolates retrieved from those specimens evaluated, 5 from 25 HIV-seropositive patients and 1 isolate from a patient whose HIV status was unknown were shown to be consistent by phenotyping and by electrophoretic karyotyping with the European reference strain of C. dubliniensis. One of the C. dubliniensis isolates had dose-dependent susceptibility to fluconazole (MIC, 16 μg/ml). These results confirm the presence of this interesting species in the United States and support the need for further investigations into the prevalence and pathogenesis of C. dubliniensis.     

Simple, Inexpensive, Reliable Method for Differentiation of Candida dubliniensis from Candida albicans

Candida dubliniensis is a recently described pathogenic species which shares many phenotypic features with Candida albicans, including the ability to form germ tubes and chlamydospores. These similarities have caused significant problems in the identification of C. dubliniensis by the average clinical mycology laboratory. To facilitate the differentiation of these species, we investigated the growth of 120 isolates of C. dubliniensis and 98 C. albicans isolates at 42 and 45°C on Emmons’ modified Sabouraud glucose agar (SGA) and 10 isolates of each species in yeast-peptone-dextrose broth. None of the C. dubliniensis isolates grew on the agar or in the broth medium at 45°C, while 11 isolates were capable of growing on SGA at 42°C. In contrast, all of the C. albicans isolates but one grew at 45°C on or in either medium. These reproducible results clearly demonstrate that the incubation of isolates suspected to be C. dubliniensis or C. albicans at 45°C provides a simple, reliable, and inexpensive method for the differentiation of the two species.     

New Assay for Measuring Cell Surface Hydrophobicities of Candida dubliniensis and Candida albicans

Hydrophobic interactions, based on cell surface hydrophobicity (CSH), are among the many and varied mechanisms of adherence deployed by the pathogenic yeast Candida albicans. Recently it was shown that, unlike C. albicans, C. dubliniensis is a species that exhibits an outer fibrillar layer consistent with constant CSH. Previously, C. dubliniensis grown at 25 or 37°C was shown to coaggregate with the oral anaerobic bacterium Fusobacterium nucleatum. C. albicans, however, demonstrated similar coaggregation only when hydrophobic or grown at 25°C. This observation implied that coaggregation of Candida cells with F. nucleatum is associated with a hydrophobic yeast cell surface. To test this hypothesis, 42 C. albicans and 40 C. dubliniensis clinical isolates, including a C. albicans hydrophobic variant, were grown at 25 and 37°C and tested with the established hydrophobicity microsphere assay, which determines CSH levels based on the number of microspheres attached to the yeast cells. The coaggregation assay was performed in parallel experiments. All C. dubliniensis isolates grown at either temperature, hydrophobic 25°C-grown C. albicans isolates, and the C. albicans hydrophobic variant, unlike the 37°C-hydrophilic C. albicans isolates, exhibited hydrophobic CSH levels with the microsphere assay and simultaneously showed maximum, 4+, coaggregation with F. nucleatum. The parallel results obtained for C. dubliniensis using both assays support the use of the CoAg assay both as a rapid assay to determine CSH and to differentiate between C. dubliniensis and C. albicans.     

Characterization of a New Clinical Yeast Species,Candida tunisiensis sp. nov., Isolated from a Strain Collection from Tunisian Hospitals

From a collection of yeast isolates isolated from patients in Tunisian hospitals between September 2006 and July 2010, the yeast strain JEY63 (CBS 12513), isolated from a 50-year-old male that suffered from oral thrush, could not be identified to the species level using conventional methods used in clinical laboratories. These methods include matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), germ tube formation, and the use of CHROMagar Candida and metabolic galleries. Sequence analysis of the nuclear rRNA (18S rRNA, 5.8S rRNA, and 26S rRNA) and internal transcribed spacer regions (ITS1 and ITS2) indicated that the ribosomal DNA sequences of this species were not yet reported. Multiple gene phylogenic analyses suggested that this isolate clustered at the base of the Dipodascaceae (Saccharomycetales, Saccharomycetes, and Ascomycota). JEY63 was named Candida tunisiensis sp. nov. according to several phenotypic criteria and its geographical origin. C. tunisiensis was able to grow at 42°C and does not form chlamydospores and hyphae but could grow as yeast and pseudohyphal forms. C. tunisiensis exhibited most probably a haploid genome with an estimated size of 10 Mb on at least three chromosomes. Using European Committee for Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) Candida albicans susceptibility breakpoints as a reference, C. tunisiensis was resistant to fluconazole (MIC = 8 μg/ml), voriconazole (MIC = 0.5 μg/ml), itraconazole (MIC = 16 μg/ml), and amphotericin B (MIC = 4 μg/ml) but still susceptible to posaconazole (MIC = 0.008 μg/ml) and caspofungin (MIC = 0.5 μg/ml). In conclusion, MALDI-TOF MS permitted the early selection of an unusual isolate, which was still unreported in molecular databases but could not be unambiguously classified based on phylogenetic approaches.     

Chromogenic Tube Test for Presumptive Identification or Confirmation of Isolates as Candida albicans

This report describes a new, modified, simple, and cost-effective method for the use of CHROMagar Candida (CHROMagar Company, Paris, France) for the presumptive identification of isolates as Candida albicans after preliminary growth. Sixty randomly selected clinical isolates were evaluated, including 38 of C. albicans. With incubation at 37°C for 24 h, the sensitivity and specificity appeared to be excellent and the test performed better than the traditional germ tube test. However, at earlier times, C. tropicalis isolates gave false-positive results.     

Prevalence of Candida dubliniensis Isolates in a Yeast Stock Collection

To establish the historical prevalence of the novel yeast species Candida dubliniensis, a survey of 2,589 yeasts originally identified as Candida albicans and maintained in a stock collection dating back to the early 1970s was undertaken. A total of 590 yeasts, including 93 (18.5%) β-glucosidase-negative isolates among 502 isolates that showed abnormal colony colors on a differential chromogenic agar and 497 other isolates, were subjected to DNA fingerprinting with the moderately repetitive sequence Ca3. On this basis, 53 yeasts were reidentified as C. dubliniensis (including the C. dubliniensis type strain, included as a blind control in the panel of yeasts). The 52 newly found isolates came from 36 different persons, and a further 3 C. dubliniensis isolates were detected by DNA fingerprinting of previously untested isolates from one of these individuals. The prevalence of C. dubliniensis among yeasts in oral and fecal samples was significantly higher than that among yeasts from other anatomical sites and was significantly higher among human immunodeficiency virus (HIV)-infected individuals than among known or presumed HIV-negative individuals. However, a single vaginal isolate and two oral isolates from healthy volunteers confirmed that the species is restricted neither to gastrointestinal sites nor to patients with overt disease. The oldest examples of C. dubliniensis were from oral samples of three patients in the United Kingdom in 1973 and 1975. In comparison with age-matched control isolates of C. albicans, the C. dubliniensis isolates showed slightly higher levels of susceptibility in vitro to amphotericin B and flucytosine and slightly lower levels of susceptibility to three azole antifungal agents.     

Echinocandin and Triazole Antifungal Susceptibility Profiles for Clinical Opportunistic Yeast and Mold Isolates Collected from 2010 to 2011: Application of New CLSI Clinical Breakpoints and Epidemiological Cutoff Values for Characterization of Geographic and Temporal Trends of Antifungal Resistance

The SENTRY Antimicrobial Surveillance Program monitors global susceptibility and resistance rates of newer and established antifungal agents. We report the echinocandin and triazole antifungal susceptibility patterns for 3,418 contemporary clinical isolates of yeasts and molds. The isolates were obtained from 98 laboratories in 34 countries during 2010 and 2011. Yeasts not presumptively identified by CHROMagar, the trehalose test, or growth at 42°C and all molds were sequence identified using internal transcribed spacer (ITS) and 28S (yeasts) or ITS, translation elongation factor (TEF), and 28S (molds) genes. Susceptibility testing was performed against 7 antifungals (anidulafungin, caspofungin, micafungin, fluconazole, itraconazole, posaconazole, and voriconazole) using CLSI methods. Rates of resistance to all agents were determined using the new CLSI clinical breakpoints and epidemiological cutoff value criteria, as appropriate. Sequencing of fks hot spots was performed for echinocandin non-wild-type (WT) strains. Isolates included 3,107 from 21 Candida spp., 146 from 9 Aspergillus spp., 84 from Cryptococcus neoformans, 40 from 23 other mold species, and 41 from 9 other yeast species. Among Candida spp., resistance to the echinocandins was low (0.0 to 1.7%). Candida albicans and Candida glabrata that were resistant to anidulafungin, caspofungin, or micafungin were shown to have fks mutations. Resistance to fluconazole was low among the isolates of C. albicans (0.4%), Candida tropicalis (1.3%), and Candida parapsilosis (2.1%); however, 8.8% of C. glabrata isolates were resistant to fluconazole. Among echinocandin-resistant C. glabrata isolates from 2011, 38% were fluconazole resistant. Voriconazole was active against all Candida spp. except C. glabrata (10.5% non-WT), whereas posaconazole showed decreased activity against C. albicans (4.4%) and Candida krusei (15.2% non-WT). All agents except for the echinocandins were active against C. neoformans, and the triazoles were active against other yeasts (MIC₉₀, 2 μg/ml). The echinocandins and triazoles were active against Aspergillus spp. (MIC₉₀/minimum effective concentration [MEC₉₀] range, 0.015 to 2 μg/ml), but the echinocandins were not active against other molds (MEC₉₀ range, 4 to >16 μg/ml). Overall, echinocandin and triazole resistance rates were low; however, the fluconazole and echinocandin coresistance among C. glabrata strains warrants continued close surveillance.     

Misidentification of Candida dubliniensis isolates with the VITEK MS

The phylogenetic relatedness of Candida dubliniensis and C. albicans may lead to misidentification of C. dubliniensis and underestimation of its clinical significance. We evaluated the performance of VITEK-MS in identifying C. dubliniensis isolates following growth on different culture media. Correct identification was documented in 98% of the isolates grown on blood agar media whereas only 44% were correctly identified from SDA or CHROMagar. The use of non-manufacturer validated media for identifying C. dubliniensis with VITEK-MS, may result in misidentification of these isolates as C. albicans. This finding calls for reassessing the accuracy of fungal isolates identification in local workflows using non-validated culture media.     

Première identification de Candida dubliniensis au centre hospitalier universitaire Ibn Rochd de Casablanca (Maroc)

Candida dubliniensis is a newly recognized yeast species with phenotypic characteristics closely related to Candida albicans and specifically identified by molecular biology. C. dubliniensis is commonly associated with oral candidiasis, especially in human immunodeficiency virus-positive patients. In this study, the authors describe the first isolates of C. dubliniensis from Morocco and compare phenotypic identification of this species with PCR. For this purpose, 12 C. albicans isolates, taken from hospitalized patients in Ibn Rochd University Hospital of Casablanca (Morocco), were selected for C. dubliniensis confirmation on the basis of their ability to produce abundant chlamydospores on Rice-Agar-Tween (RAT) medium and absence of growth at 45 °C. Presumptive identification of C. dubliniensis was performed using different phenotypic methods: ability to produce chlamydospores on casein agar medium, carbohydrate assimilation profiles and latex agglutination. Final identification was confirmed at the CRMA, Pasteur Institute of Paris, by positive amplification with C. dubliniensis specific primers. Thus, five isolates were confirmed in this study as C. dubliniensis, two were taken from oropharyngeal samples of HIV infected patients, while the remaining three were taken from bronchial aspirations of non-HIV patients.     

Epidemiology,Antifungal Susceptibility,and Pathogenicity of Candida africana Isolates from the United Kingdom

Candida africana was previously proposed as a new species within the Candida albicans species complex, together with C. albicans and C. dubliniensis, although further phylogenetic analyses better support its status as an unusual variant within C. albicans. Here we show that C. africana can be distinguished from C. albicans and C. dubliniensis by pyrosequencing of a short region of ITS2, and we have evaluated its occurrence in clinical samples by pyrosequencing all presumptive isolates of C. albicans submitted to the Mycology Reference Laboratory over a 9-month period. The C. albicans complex constituted 826/1,839 (44.9%) of yeast isolates received over the study period and included 783 isolates of C. albicans, 28 isolates of C. dubliniensis, and 15 isolates of C. africana. In agreement with previous reports, C. africana was isolated exclusively from genital specimens, in women in the 18-to-35-year age group. Indeed, C. africana constituted 15/251 (6%) of “C. albicans” isolates from female genital specimens during the study period. C. africana isolates were germ tube positive, grew significantly more slowly than C. albicans and C. dubliniensis on conventional mycological media, could be distinguished from the other members of the C. albicans complex by appearance on chromogenic agar, and were incapable of forming chlamydospores. Here we present the detailed evaluation of epidemiological, phenotypic, and clinical features and antifungal susceptibility profiles of United Kingdom isolates of C. africana. Furthermore, we demonstrate that C. africana is significantly less pathogenic than C. albicans and C. dubliniensis in the Galleria mellonella insect systemic infection model.     

International Surveillance of Bloodstream Infections Due to Candida Species: Frequency of Occurrence and Antifungal Susceptibilities of Isolates Collected in 1997 in the United States, Canada, and South America for the SENTRY Program

An international program of surveillance of bloodstream infections (BSIs) in the United States, Canada, and South America between January and December 1997 detected 306 episodes of candidemia in 34 medical centers (22 in the United States, 6 in Canada, and 6 in South America). Eighty percent of the BSIs were nosocomial and 50% occurred in patients hospitalized in an intensive care unit. Overall, 53.3% of the BSIs were due to Candida albicans, 15.7% were due to C. parapsilosis, 15.0% were due to C. glabrata, 7.8% were due to C. tropicalis, 2.0% were due to C. krusei, 0.7% were due to C. guilliermondii, and 5.8% were due to Candida spp. However, the distribution of species varied markedly by country. In the United States, 43.8% of BSIs were due to non-C. albicans species. C. glabrata was the most common non-C. albicans species in the United States. The proportion of non-C. albicans BSIs was slightly higher in Canada (47.5%), where C. parapsilosis, not C. glabrata, was the most common non-C. albicans species. C. albicans accounted for 40.5% of all BSIs in South America, followed by C. parapsilosis (38.1%) and C. tropicalis (11.9%). Only one BSI due to C. glabrata was observed in South American hospitals. Among the different species of Candida, resistance to fluconazole (MIC, ≥64 μg/ml) and itraconazole (MIC, ≥1.0 μg/ml) was observed with C. glabrata and C. krusei and was observed more rarely among other species. Isolates of C. albicans, C. parapsilosis, C. tropicalis, and C. guilliermondii were all highly susceptible to both fluconazole (99.4 to 100% susceptibility) and itraconazole (95.8 to 100% susceptibility). In contrast, 8.7% of C. glabrata isolates (MIC at which 90% of isolates are inhibited [MIC₉₀], 32 μg/ml) and 100% of C. krusei isolates were resistant to fluconazole, and 36.9% of C. glabrata isolates (MIC₉₀, 2.0 μg/ml) and 66.6% of C. krusei isolates were resistant to itraconazole. Within each species there were no geographic differences in susceptibility to fluconazole or itraconazole.     

Hypertonic Xylose Agar Medium: A Novel Medium for Differentiation of Candida dubliniensis from Candida albicans

Background: Candida dubliniensis is a pathogenic Candida species which shares many phenotypic features with Candida albicans. These similarities have caused significant problems in the identification of C. dubliniensis in an average clinical mycology laboratory. Several phenotypic-based tests have been developed to distinguish C. albicans from C. dubliniensis but none has been demonstrated being sufficient alone for accurate differentiation of the two species. Aim: To facilitate the differentiation of these species, we evaluated the utility of a novel medium ‘Hypertonic Xylose Agar Medium’ (HXAM). Materials and Methods: A total of 200 Candida spp. were tested in this study which included 186 stock strains of C. albicans and 14 strains of C. dubliniensis. Identification of all these strains was confirmed by polymerase chain reaction-restriction fragment length polymorphism using BlnI (AvrII) enzyme. All isolates were inoculated on HXAM, incubated at 28°C and examined for visible growth every day up to 7 days. Results: On this medium at 28°C, all 186 C. albicans isolates showed visible growth at 48 h of incubation whereas none of the 14 C. dubliniensis isolates did so even on extending the incubation period up to 7 days. Conclusion: Hence, we propose HXAM as a sole phenotypic method for identifying C. dubliniensis from germ-tube-positive isolates or from stock collections of known C. albicans.     

Constant Low Rate of Fungemia in Norway, 1991 to 1996

Since 1991 information on yeast isolates from blood cultures has been recorded prospectively from all microbiological laboratories (5 university and 16 county or local hospital laboratories) in Norway (population, 4.3 million). From 1991 to 1996 a total of 571 episodes of fungemia in 552 patients occurred (1991, 109 episodes; 1992, 81 episodes; 1993, 93 episodes; 1994, 89 episodes; 1995, 98 episodes; and 1996, 101 episodes). The fungemia rates per 10,000 patient days were 0.29 in 1991 and 0.27 in 1996. The average rates for the years 1991 to 1996 were 0.37 for the university laboratories and 0.20 for the other laboratories. These rates are low compared to the rate (0.76) in five Dutch university hospitals in 1995 and the rate (2.0) in Iowa in 1991. The four most frequently isolated species were Candida albicans (66%), Candida glabrata (12.5%), Candida parapsilosis (7.6%), and Candida tropicalis (6.4%). The incidences of both C. albicans (range, 63 to 73%) and C. glabrata (range, 8.4 to 15.7%) varied somewhat throughout this period, but no significant increase or decrease was noted. MICs of amphotericin B, flucytosine, and fluconazole were determined for 89% of the isolates. All were susceptible to amphotericin B, and only 29 (5.6%) strains had decreased susceptibility to flucytosine. All C. albicans isolates were susceptible to fluconazole. The percentage of yeast isolates with decreased susceptibility to fluconazole (MICs, ≥16 μg/ml) did increase, from 9.6% in 1991 and 1992 to 12.2% in 1994, 16.1% in 1995, and 18.6% in 1996. This was largely due to increases in the percentages of resistant C. glabrata and Candida krusei strains in the last 2 years. Compared to the incidence in other countries, it is remarkable that Norway has such a low and constant incidence of fungemia. A possible reason for this difference might be a restricted antibiotic use policy in Norway.     

Multidrug-Resistant Candida auris Misidentified as Candida haemulonii: Characterization by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and DNA Sequencing and Its Antifungal Susceptibility Profile Variability by Vitek 2, CLSI Broth Microdilution,and Etest Method

Candida auris is a multidrug-resistant yeast that causes a wide spectrum of infections, especially in intensive care settings. We investigated C. auris prevalence among 102 clinical isolates previously identified as Candida haemulonii or Candida famata by the Vitek 2 system. Internal transcribed spacer region (ITS) sequencing confirmed 88.2% of the isolates as C. auris, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) easily separated all related species, viz., C. auris (n = 90), C. haemulonii (n = 6), C. haemulonii var. vulnera (n = 1), and Candida duobushaemulonii (n = 5). The in vitro antifungal susceptibility was determined using CLSI broth microdilution (CLSI-BMD), the Vitek 2 antifungal susceptibility test, and the Etest method. C. auris isolates revealed uniformly elevated fluconazole MICs (MIC₅₀, 64 μg/ml), and an alarming percentage of isolates (37%) exhibited elevated caspofungin MICs by CLSI-BMD. Notably, 34% of C. auris isolates had coexisting elevated MICs (≥2 μg/ml) for both fluconazole and voriconazole, and 10% of the isolates had elevated coexisting MICs (≥2 μg/ml) to two additional azoles, i.e., posaconazole and isavuconazole. In contrast to reduced amphotericin B MICs by CLSI-BMD (MIC₅₀, 1 μg/ml) for C. auris, elevated MICs were noted by Vitek 2 (MIC₅₀, 8 μg/ml), which were statistically significant. Candida auris remains an unnoticed pathogen in routine microbiology laboratories, as 90% of the isolates characterized by commercial identification systems are misidentified as C. haemulonii. MALDI-TOF MS proved to be a more robust diagnostic technique for rapid identification of C. auris. Considering that misleading elevated MICs of amphotericin B by the Vitek AST-YS07 card may lead to the selection of inappropriate therapy, a cautionary approach is recommended for laboratories relying on commercial systems for identification and antifungal susceptibility testing of rare yeasts.     

Comparative Evaluation of National Committee for Clinical Laboratory Standards Broth Macrodilution and Agar Dilution Screening Methods for Testing Fluconazole Susceptibility of Cryptococcus neoformans

A simple screening method for fluconazole susceptibility of Cryptococcus neoformans using 2% dextrose Sabouraud dextrose agar (SabDex) with fluconazole was compared to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method. By this method, fluconazole-susceptible C. neoformans isolates are significantly smaller on medium with fluconazole than on fluconazole-free medium. Isolates with decreased susceptibility have normal-size colonies on medium containing fluconazole. The 48-h NCCLS broth macrodilution MICs (NCCLS MICs) for isolates with normal-size colonies on 8- or 16-μg/ml fluconazole plates were predicted to be ≥8 or ≥16 μg/ml, respectively. On medium with 16 μg of fluconazole per ml, all strains (84 of 84) for which the NCCLS MICs were <16 μg/ml were correctly predicted, as were all isolates (7 of 7) for which the MICs were ≥16 μg/ml. Agar dilution appears to be an effective screening method for fluconazole resistance in C. neoformans.     

Multilocus sequence typing reveals that the population structure of Candida dubliniensis is significantly less divergent than that of Candida albicans

The pathogenic yeast Candida dubliniensis is phylogenetically very closely related to Candida albicans, and both species share many phenotypic and genetic characteristics. DNA fingerprinting using the species-specific probe Cd25 and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal gene cluster previously showed that C. dubliniensis is comprised of three major clades comprising four distinct ITS genotypes. Multilocus sequence typing (MLST) has been shown to be very useful for investigating the epidemiology and population biology of C. albicans and has identified many distinct major and minor clades. In the present study, we used MLST to investigate the population structure of C. dubliniensis for the first time. Combinations of 10 loci previously tested for MLST analysis of C. albicans were assessed for their discriminatory ability with 50 epidemiologically unrelated C. dubliniensis isolates from diverse geographic locations, including representative isolates from the previously identified three Cd25-defined major clades and the four ITS genotypes. Dendrograms created by using the unweighted pair group method with arithmetic averages that were generated using the data from all 10 loci revealed a population structure which supports that previously suggested by DNA fingerprinting and ITS genotyping. The MLST data revealed significantly less divergence within the C. dubliniensis population examined than within the C. albicans population. These findings show that MLST can be used as an informative alternative strategy for investigating the population structure of C. dubliniensis. On the basis of the highest number of genotypes per variable base, we recommend the following eight loci for MLST analysis of C. dubliniensis: CdAAT1b, CdACC1, CdADP1, CdMPIb, CdRPN2, CdSYA1, exCdVPS13, and exCdZWF1b, where “Cd” indicates C. dubliniensis and “ex” indicates extended sequence.     

Antifungal Susceptibility Testing of Candida Isolates from the Candida Surveillance Study

Candida species are a common cause of nosocomial bloodstream infections. Recent surveillance has shown an increase in the relative proportion of infections caused by Candida glabrata, which has reduced susceptibility to fluconazole. We undertook sentinel surveillance with antifungal susceptibility testing to monitor the trends in the proportions of various Candida species causing invasive disease. Forty-one institutions participated in the Candida Surveillance Study. All isolates were submitted to a central laboratory for identification and susceptibility testing. Susceptibility testing was performed in compliance with CLSI guidelines using a custom, broth dilution, microtiter system. There were 5,900 isolates submitted for identification and antifungal susceptibility testing. The distribution of species was as follows: C. albicans, 2,567 (43.5%) isolates; C. glabrata, 1,464 (24.8%) isolates; C. parapsilosis, 1,048 (17.8%) isolates; C. tropicalis, 527 (8.9%) isolates; C. krusei, 109 (1.9%) isolates; C. lusitaniae, 76 (1.3%) isolates; and other Candida species, 109 (1.9%) isolates. Resistance to fluconazole occurred in 1.2% of C. albicans isolates, 5.9% of C. glabrata isolates, 0.3% of C. parapsilosis isolates, and 0.4% of C. tropicalis isolates. Resistance to fluconazole was highly predictive of resistance to voriconazole. Resistance to echinocandins was rarely found, occurring in only 0.2% of all isolates. The rate of fluconazole susceptibility increased significantly from 87.5% in 2005 to 97.4% in 2007. The proportion of cases of disease caused by various Candida species did not change appreciably between 2004 and 2007, and the rate of antifungal susceptibility was high.Over the last 20 years, Candida species have become prominent nosocomial pathogens (1, 2, 21). Over the past decade, reports have documented a shift away from Candida albicans as the cause of the majority of invasive infections toward non-C. albicans species (7, 8, 19). Candida glabrata, which is less susceptible to fluconazole, is the species whose incidence has increased the most to account for the decrease in the proportion of cases of invasive disease caused by C. albicans (7, 8, 19, 21). The Centers for Disease Control and Prevention (CDC) conducted population-based surveillance for Candida bloodstream infections over two different time periods: in 1992 and 1993 and from 1998 to 2000 (7, 8). During the first surveillance period, C. albicans accounted for 52% of the isolates and C. glabrata accounted for 12%. During the second surveillance period, C. albicans accounted for 45% and C. glabrata accounted for 24%. During the latter surveillance period, when the activity of fluconazole was tested, the MIC₅₀ and MIC₉₀ were as follows: for C. albicans, ≤0.125 μg/ml and 0.5 μg/ml, respectively, and for C. glabrata, 4 μg/ml and 16 μg/ml, respectively (7, 8). In another sentinel surveillance study conducted from 1992 to 2001, the proportion of cases of disease caused by C. glabrata was only 18%, but the proportion did increase over the surveillance period in the United States (18). Contrary to the findings from the CDC surveillance, the other sentinel surveillance study found that the proportion of C. glabrata isolates which were susceptible to fluconazole increased from 15% in 1992 to 64% in 2001 (18). However, additional sentinel surveillance conducted by the same group between 1997 and 2005 did not show a significant shift in the proportion of cases of disease caused by C. glabrata (14). Additionally, that study did not detect any significant change in the rate of fluconazole resistance (14).In order to monitor changing trends in the species distribution and antifungal susceptibility patterns of invasive Candida isolates, we undertook a sentinel surveillance program involving a variety of community and academic medical institutions in the United States.     

Candida bracarensis detected among isolates of Candida glabrata by peptide nucleic acid fluorescence in situ hybridization: susceptibility data and documentation of presumed infection

Molecular taxonomic studies have revealed new Candida species among phenotypically delineated species, the best example being Candida dubliniensis. This study was designed to determine the occurrence of two new molecularly defined species, Candida bracarensis and Candida nivariensis, which are closely related to and identified as Candida glabrata by phenotypic assays. A total of 137 recent clinical isolates of C. glabrata identified by phenotypic characteristics was tested with C. bracarensis and C. nivariensis species-specific peptide nucleic acid fluorescence in situ hybridization probes. Three of 137 (2.2%) isolates were positive with the C. bracarensis probe, whereas the control strain, but none of the clinical isolates, was positive with the C. nivariensis probe. D1/D2 sequencing confirmed the identification of the three isolates as representing C. bracarensis. Clinically, one C. bracarensis isolate was recovered from a presumed infection, a polymicrobial pelvic abscess in a patient with perforated diverticulitis. The other two isolates were recovered from two adult oncology patients who were only colonized. C. bracarensis was white on CHROMagar Candida, had variable API-20C patterns that overlapped with C. nivariensis and some C. glabrata isolates, and had variable results with a rapid trehalose assay. Interestingly, an isolate from one of the colonized oncology patients was resistant to fluconazole, itraconazole, voriconazole, and posaconazole in vitro. In summary, C. bracarensis was detected among clinical isolates of C. glabrata, while C. nivariensis was not. One C. bracarensis isolate causing a presumed deep infection was recovered, and another isolate was azole resistant. Whether clinical laboratories should identify C. bracarensis will require more data.     

Fluconazole Disk Diffusion Susceptibility Testing of Candida Species

We describe a simple procedure for detecting fluconazole-resistant yeasts by a disk diffusion method. Forty clinical Candida sp. isolates were tested on RPMI-glucose agar with either 25- or 50-μg fluconazole disks. With 25-μg disks, zones of inhibition of ≥20 mm at 24 h accurately identified 29 of 29 isolates for which MICs were ≤8 μg/ml, and with 50-μg disks, zones of ≥27 mm identified 28 of 29 such isolates. All 11 isolates for which MICs were >8 μg/ml were identified by using either disk. Disk diffusion may be a useful screening method for clinical microbiology laboratories.     

In Vitro Activities of Isavuconazole and Comparator Antifungal Agents Tested against a Global Collection of Opportunistic Yeasts and Molds

Isavuconazole is a new broad-spectrum triazole with a favorable pharmacokinetic and safety profile. We report the MIC distributions for isavuconazole and 111 isolates of Candida (42 Candida albicans, 25 Candida glabrata, 22 Candida parapsilosis, 14 Candida tropicalis, and 8 Candida krusei isolates), as determined by Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution (BMD) methods. Also, the relative activities of isavuconazole, itraconazole, fluconazole, posaconazole, voriconazole, and the three echinocandins were assessed against a recent (2011) global collection of 1,358 isolates of Candida spp., 101 of Aspergillus spp., 54 of non-Candida yeasts, and 21 of non-Aspergillus molds using CLSI BMD methods. The overall essential agreement (EA) (±2 log₂ dilutions) between the CLSI and EUCAST methods was 99.1% (EA at ±1 log₂ dilution, 90.1% [range, 80.0 to 100.0%]). The activities of isavuconazole against the larger collection of Candida spp. and Aspergillus spp. were comparable to those of posaconazole and voriconazole; the MIC₉₀ values for isavuconazole, posaconazole, and voriconazole against Candida spp. were 0.5, 1, and 0.25 μg/ml and against Aspergillus spp. were 2, 1, and 1 μg/ml, respectively. Isavuconazole showed good activities against Cryptococcus neoformans (MIC₉₀, 0.12 μg/ml) and other non-Candida yeasts (MIC₉₀, 1 μg/ml) but was less potent against non-Aspergillus molds (MIC₉₀, >8 μg/ml). Isavuconazole MIC values for three mucormycete isolates were 4, 1, and 2 μg/ml, whereas all three were inhibited by 1 μg/ml posaconazole. Isavuconazole demonstrates broad-spectrum activity against this global collection of opportunistic fungi, and the CLSI and EUCAST methods can be used to test this agent against Candida, with highly comparable results.