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Background: Candida auris is a yeast that is causing nosocomial outbreaks in healthcare facilities around the world. There is a risk of the misidentification of C. auris with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-when libraries are used that lack C. auris spectra, or when conventional biochemical methods are used., Methods: We conducted an external quality assessment to evaluate the ability of Dutch clinical microbiological laboratories to identify C. auris , and to raise awareness about the risk of misidentification., Results: 35/47 participating laboratories were able to identify C. auris correctly. Only 2/14 labs that potentially misidentified C. auris with their primary identification methods specified that they would perform additional tests to exclude C. auris when appropriate. 45/47 labs used MALDI-TOF MS systems to identify Candida species., Conclusions: There was a lack of awareness about the potential misidentification of C. auris in many labs that used MALDI-TOF MS with libraries that lacked C. auris spectra, and labs that used Vitek 2. However, as the currently available MALDI-TOF MS libraries in The Netherlands contain several C. auris spectra, we expect that currently almost all participating laboratories are able to identify C. auris correctly, as 45/47 participating laboratories use MALDI-TOF MS as their primary yeast identification method.

The in vitro susceptibilities of 24 molecularly identified dimorphic fungi belonging to the genera Adiaspiromyces , Blastomyces , and Emergomyces within the family Ajellomycetaceae were tested against 8 standard antifungal agents using CLSI document M38-A2. Amphotericin B and posaconazole had the lowest geometric mean MICs (<0.05 μg/ml) followed by itraconazole (<0.07 μg/ml), voriconazole (<0.15 μg/ml), and isavuconazole (<0.42 μg/ml) while fluconazole was not active. Micafungin demonstrated good in vitro antifungal activity against Emergomyces (geometric mean minimum effective concentration [GM MEC] 0.1 μg/ml) and Blastomyces (GM MEC <0.017 μg/ml)., (Copyright © 2017 American Society for Microbiology.)

In vitro susceptibility testing of Fusarium is becoming increasingly important because of frequency and diversity of infections and because resistance profiles are species-specific. Reference methods for antifungal susceptibility testing (AFST) are those of Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility (EUCAST), but breakpoints (BPs) have not yet been established. One of the problems is that phylogenetic distances between Fusarium species are much smaller than between species of, e.g., Candida . Epidemiological cutoff values (ECVs) for some Fusarium species have been determined in order to differentiate wild-type from non-wild-type isolates. In clinical routine, commercially available assays such as Etest, Sensititre or others provide essential agreement with reference methods. Our objective is to summarize antifungal susceptibility testing of Fusarium genus in the clinical laboratory: how to do it, when to do it, and how to interpret it.

The in vitro susceptibilities of 1,099 molecularly identified clinical Candida isolates against 8 antifungal drugs were determined using the EUCAST microdilution method. A new simple, objective, and mathematically solid method for determining epidemiological cutoff values (ECOFFs) was developed by derivatizing the MIC distribution and determining the derivatized ECOFF (dECOFF) as the highest MIC with the maximum second derivative. The dECOFFs were similar (95% agreement within 1 dilution) to the EUCAST ECOFFs. Overall, low non-wild-type/resistance rates were found. The highest rates were found for azoles with C. parapsilosis (2.7 to 9.8%), C. albicans (7%), and C. glabrata (1.7 to 2.3%) and for echinocandins with C. krusei (3.3%), C. albicans (1%), and C. tropicalis (1.7%)., (Copyright © 2017 American Society for Microbiology.)

Ecthyma gangrenosum (EG) involves necrotic cutaneous lesions caused by bacteria, mainly Pseudomonas aeruginosa, and is usually seen in immunocompromised patients with septicemia. However, clinically similar infections have been published with fungi as etiologic agents. We present a case of an EG-like lesion due to Fusarium oxysporum confirmed by clinical diagnosis, culture and molecular identification and discuss the definition of EG., Competing Interests: The authors declare that they have no competing interests.

The emergence of resistant strains among common and rare Candida species necessitates continuous monitoring of the in vitro susceptibilities of those isolates. We therefore assessed the in vitro activities of micafungin against 1,099 molecularly identified isolates belonging to 5 common and 20 rare Candida species by the EUCAST, CLSI, and Etest methods, assessing both the intralaboratory agreement and the interlaboratory agreement for two centers. The median micafungin EUCAST MICs were as follows, from the lowest to the highest: for Candida albicans, 0.004 mg/liter; for C. glabrata, 0.016 mg/liter; for C. tropicalis, 0.031 mg/liter; for C. krusei, 0.125 mg/liter; for C. parapsilosis, 2 mg/liter. Among rare Candida species, high MICs were found for C. guilliermondii, C. lipolytica, C. orthopsilosis, C. metapsilosis, and C. fermentati. No resistant isolates were found by the CLSI method, whereas resistance rates of 1 to 2% were found by the EUCAST method. Overall, the EUCAST method resulted in MICs 1 to 2 dilutions higher than those found by the CLSI and Etest methods. The intra- and interlaboratory agreement between methods was >92%, except for the interlaboratory agreement between the EUCAST and CLSI methods (81%), where 17 to 31% of the differences were >2 2-fold dilutions for C. albicans, C. glabrata, C. tropicalis, and other rare Candida species and <6% for C. parapsilosis and C. krusei For the other interlaboratory comparisons, the EUCAST method resulted in higher MICs than the Etest method for all species, but <7% of these differences were >2 2-fold dilutions. Overall, the CLSI method resulted in lower MICs than the Etest method, with 11% of all isolates demonstrating >2 2-fold-dilution differences (6 to 20% for C. albicans, C. tropicalis, and rare Candida species; <5% for C. glabrata, C. krusei, and C. parapsilosis) and smaller differences found after 24 h. Despite these differences, categorical agreement was excellent (>97%), with only 1 to 2% very major errors between the EUCAST method and the other two methods., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: Fusarium species cause a broad spectrum of infections, from superficial to disseminated disease. Because Fusarium species are intrinsically resistant to most antifungal drugs, new approaches are needed. The aim of the present study was to evaluate the in vitro combination of natamycin with currently used antifungal drugs., Methods: The in vitro interactions of combinations between natamycin and voriconazole, itraconazole and micafungin applied to 20 clinical Fusarium strains (members of Fusarium falciforme, Fusarium napiforme, Fusarium petroliphilum, Fusarium proliferatum, Fusarium pseudensiforme and Fusarium sacchari) were evaluated using a chequerboard microdilution method. The MICs of all drugs alone and in combination were determined visually after 48 h and interactions were assessed using fractional inhibitory concentration index (FICI) analysis., Results: MICs of voriconazole and natamycin alone were 4 to >16 and 4-8 mg/L, respectively. Values were reduced 3.5-10-fold to 0.02-0.5 mg/L and 0.5-5-fold to 0.13-2 mg/L in combination, for the currently used antifungals and natamycin, respectively, demonstrating additive to synergistic interactions. The combinations natamycin/voriconazole, natamycin/itraconazole and natamycin/micafungin were synergistic (FICI ≤0.5) for 70%, 15% and 5% of the strains, respectively. No antagonism was found., Conclusions: The combination of natamycin with voriconazole was strongly synergistic at clinically achievable serum concentrations., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The in vitro susceptibilities of 24 worldwide Exserohilum isolates belonging to 10 species from human and environmental sources were determined for eight antifungal drugs. The strains were characterized by internal transcribed spacer (ITS) sequencing and amplified fragment length polymorphism fingerprinting. Posaconazole had the lowest geometric mean MIC (0.16 μg/ml), followed by micafungin (0.21 μg/ml), amphotericin B (0.24 μg/ml), itraconazole (0.33 μg/ml), voriconazole (0.8 μg/ml), caspofungin (1.05 μg/ml), isavuconazole (1.38 μg/ml), and fluconazole (15.6 μg/ml)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Objectives: The aim of the present study was to evaluate and assess the in vitro activity of eight drugs, including the new azole isavuconazole, against 81 strains representing 13 species of the Fusarium fujikuroi species complex., Methods: A total of 81 Fusarium spp. isolates, within the F. fujikuroi species complex, were identified by molecular methods and tested according to CLSI M38-A2. Eight antifungal compounds, including the new azole isavuconazole, were tested. Isolates were selected to represent the widest variety of geographical regions and to include clinical as well as environmental strains., Results: Susceptibility profiles differed between and within species, with Fusarium verticillioides showing the lowest MICs and Fusarium nygamai the highest MICs. Amphotericin B was the most active drug, followed by voriconazole, posaconazole, isavuconazole and natamycin. The remaining antifungals (fluconazole, itraconazole and micafungin) showed poor activity with MIC/minimum effective concentration values of ≥ 32, ≥ 16 and >8 mg/L, respectively., Conclusions: Resistance patterns in the F. fujikuroi species complex are species specific and therefore identification down to species level is important for the choice of antifungal treatment., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Members of the Fusarium solani species complex (FSSC) are causing the majority of the fusariosis in humans. Disseminated fusariosis has a high mortality and is predominantly observed in patients with leukemia. Here, we present the case of a fatal infection by a Fusarium strain with a degenerated phenotype, in a patient with acute lymphatic leukemia. Multiple nasal and skin biopsies as well as blood cultures yielded fungal growth, while in direct and histopathological examination of biopsy material septate hyphae were visible. Initial colonies were white with slimy masses with microconidia reminiscent of Fusarium/Acremonium, but with conidiospore production directly on the hyphae. Multi-locus sequence typing discerned a pionnotal-morphologically degenerated-colony of the recently recognized F. petroliphilum as etiological agent. The culture returned to a typical F. solani species complex morphology only after several weeks of growth in culture. Antifungal susceptibility tests indicate amphotericin B as best drug for this FSSC member rather than any of the azoles or echinocandins.

The in vitro activities of eight antifungal drugs against 106 clinical and environmental isolates of waterborne and cutaneous Exophiala species were tested. The MICs and minimum effective concentrations for 90% of the strains tested (n = 106) were, in increasing order, as follows: posaconazole, 0.063 μg/ml; itraconazole, 0.25 μg/ml; micafungin, 1 μg/ml; voriconazole, 2 μg/ml; isavuconazole, 4 μg/ml; caspofungin, 8 μg/ml; amphotericin B, 16 μg/ml; fluconazole, 64 μg/ml.

A global collection of Cladophialophora carrionii strains (n = 81) was tested against nine antifungal drugs. MIC90s of all strains were as follows in increasing order: itraconazole and posaconazole, 0.063 μg/ml; terbinafine, 0.125 μg/ml; isavuconazole and voriconazole, 0.25 μg/ml; caspofungin, 2 μg/ml; micafungin, 4 μg/ml; amphotericin B, 8 μg/ml; and fluconazole, 64 μg/ml.

Currently, therapy of black-grain mycetoma caused by Madurella mycetomatis consists of extensive debridement of the infected tissue combined with prolonged antifungal therapy with ketoconazole or itraconazole. In the present study, the in vitro activity of the new triazole isavuconazole toward M. mycetomatis was evaluated. Isavuconazole appeared to have high activity against M. mycetomatis, with MICs ranging from ≤0.016 to 0.125 μg/ml. Due to its favorable pharmacokinetics, isavuconazole could be a promising antifungal agent in the treatment of mycetoma.

A set of 300 Dutch Cryptococcus neoformans isolates, obtained from 237 patients during 1977 to 2007, was investigated by determining the mating type, serotype, and AFLP and microsatellite genotype and susceptibility to seven antifungal compounds. Almost half of the studied cases were from HIV-infected patients, followed by a patient group of individuals with other underlying diseases and immunocompetent individuals. The majority of the isolates were mating type α and serotype A, followed by αD isolates and other minor categories. The most frequently observed genotype was AFLP1, distantly followed by AFLP2 and AFLP3. Microsatellite typing revealed a high genetic diversity among serotype A isolates but a lower diversity within the serotype D set of isolates. One patient was infected by multiple AFLP genotypes. Fluconazole and flucytosine had the highest geometric mean MICs of 2.9 and 3.5 μg/ml, respectively, while amphotericin B (0.24 μg/ml), itraconazole (0.08 μg/ml), voriconazole (0.07 μg/ml), posaconazole (0.06 μg/ml), and isavuconazole (0.03 μg/ml) had much lower geometric mean MICs. One isolate had a high flucytosine MIC (>64 μg/ml), while decreased susceptibility (≥16 μg/ml) for flucytosine and fluconazole was found in 9 and 10 C. neoformans isolates, respectively.

Since the separation of Pseudallescheria boydii and P. apiosperma in 2010, limited data on species-specific susceptibility patterns of these and other species of Pseudallescheria and its anamorph Scedosporium have been reported. This study presents the antifungal susceptibility patterns of members affiliated with both entities. Clinical and environmental isolates (n = 332) from a wide range of sources and origins were identified down to species level and tested according to CLSI M38-A2 against eight antifungal compounds. Whereas P. apiosperma (geometric mean MIC/minimal effective concentration [MEC] values of 0.9, 2.4, 7.4, 16.2, 0.2, 0.8, 1.5, and 6.8 μg/ml for voriconazole, posaconazole, isavuconazole, itraconazole, micafungin, anidulafungin, caspofungin, and amphotericin B, respectively) and P. boydii (geometric mean MIC/MEC values of 0.7, 1.3, 5.7, 13.8, 0.5, 1.4, 2.3, and 11.8 μg/ml for voriconazole, posaconazole, isavuconazole, itraconazole, micafungin, anidulafungin, caspofungin, and amphotericin B, respectively) had similar susceptibility patterns, those for S. aurantiacum, S. prolificans, and S. dehoogii were different from each other. Voriconazole was the only drug with significant activity against S. aurantiacum isolates. The MIC distributions of all drugs except voriconazole did not show a normal distribution and often showed two subpopulations, making a species-based prediction of antifungal susceptibility difficult. Therefore, antifungal susceptibility testing of all clinical isolates remains essential for targeted antifungal therapy. Voriconazole was the only compound with low MIC values (MIC(90) of ≤ 2 μg/ml) for P. apiosperma and P. boydii. Micafungin and posaconazole showed moderate activity against the majority of Scedosporium strains.

Background: Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries., Methodology/principal Findings: Genetic diversity of Asian isolates of C. neoformans was determined using microsatellite analysis with nine microsatellite markers. The analysis revealed eight microsatellite complexes (MCs) which showed different distributions among geographically defined populations. A correlation between MCs and HIV-status was observed. Microsatellite complex 2 was mainly associated with isolates from HIV-negative patients, whereas MC8 was associated with those from HIV-positive patients. Most isolates were susceptible to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, but 17 (3.4%) and 10 (2%) were found to be resistant to 5-flucytosine and fluconazole, respectively. Importantly, five Indonesian isolates (approximately 12.5% from all Indonesian isolates investigated and 1% from the total studied isolates) were resistant to both antifungals. The majority of 5-flucytosine resistant isolates belonged to MC17., Conclusions: The findings showed a different distribution of genotypes of C. neoformans var. grubii isolates from various countries in Asia, as well as a correlation of the microsatellite genotypes with the original source of the strains and resistance to 5-flucytosine.

Apophysomyces elegans is an emerging pathogen in India. We planned the present study to analyze the clinical pattern of the disease, to perform molecular strain typing, and to determine the in vitro activities of eight antifungal drugs against A. elegans. A total of 16 clinical and two environmental A. elegans isolates were included in the study. The clinical histories of the patients were noted. MICs or minimum effective concentrations (MECs) were determined for antifungal drugs by microdilution testing in accordance with CLSI standard M38-A2 guidelines. Of 16 patients, seven had rhino-cerebral, five had cutaneous, and three had renal zygomycosis. One patient had osteomyelitis. Uncontrolled diabetes was observed in 63% of the patients. Amplified fragment length polymorphism (AFLP) analysis divided the strains into two clearly different clades. The fingerprints of the environmental strains (including the type strain) were clearly different from those of the clinical strains. The MIC50s and MIC90s for amphotericin B, itraconazole, posaconazole, and isavuconazole were 2 and 4, 1 and 2, 0.5 and 1, and 2 and 4 μg/ml, respectively. The strains had high MICs for fluconazole, voriconazole, and echinocandins. The study indicates a possible change in the clinical pattern of zygomycosis due to A. elegans in India. The fungus caused not only cutaneous or subcutaneous infection but also other deep-seated infections, and the disease is commonly associated with uncontrolled diabetes. The AFLP patterns show a clear difference between environmental and clinical strains. Posaconazole is the most active drug against the isolates, followed by itraconazole. The MICs of amphotericin B against A. elegans were higher than those of the other drugs.

The amplified fragment length polymorphism technique has been applied to identify neurotropic chaetothyrialean black yeasts and relatives from clinical sources. Cladophialophora bantiana, C. emmonsii, C. arxii, C. devriesii, and C. modesta, previously identified on the basis of sequencing and phenotypic and physiological criteria, were confirmed by cluster analysis, demonstrating the clear separation of C. bantiana as a rather homogeneous group from the other species. C. bantiana is a neurotropic fungus causing cerebral abscesses with a mortality of up to 70%. Successful therapy consists of neurosurgical intervention and optimal antifungal therapy. Since the latter is not clearly defined in a large series, we tested the in vitro activities of eight antifungal drugs against clinical isolates of C. bantiana (n = 37), C. modesta (n = 2), C. arxii (n = 1), C. emmonsii (n = 1), and C. devriesii (n = 1), all of which had caused invasive infections. The resulting MIC(90)s for all neurotropic C. bantiana strains were as follows, in increasing order: posaconazole, 0.125 microg/ml; itraconazole, 0.125 microg/ml; isavuconazole, 0.5 microg/ml; amphotericin B, 1 microg/ml; voriconazole, 2 microg/ml; anidulafungin, 2 microg/ml; caspofungin, 4 microg/ml; and fluconazole, 64 microg/ml. On the basis of these in vitro results and the findings of previous clinical and animal studies, posaconazole seems to be a good alternative to the standard treatment, amphotericin B, for C. bantiana cerebral infections. The new agent isavuconazole, which is also available as an intravenous preparation, has adequate activity against C. bantiana.

Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid leukemia. Sequence analysis revealed the isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp. nov., with CBS 10761(T) as the holotype strain, belonging to the Ovoides clade. In the D1-D2 large-subunit rRNA gene analysis, T. dohaense is a sister species to T. coremiiforme, and in the internal transcribed spacer analysis, the species is basal to the other species of this clade. Molecular identification of the strains yielded 17 T. asahii, 3 T. inkin, 2 T. japonicum, 2 T. faecale, and 3 T. dohaense isolates. The former four species exhibited low MICs for five antifungal azoles but showed high MICs for amphotericin B. T. dohaense demonstrated the lowest amphotericin B MIC (1 mg/liter). For the majority of T. asahii isolates, amphotericin B MICs were high (MIC at which 90% of isolates were inhibited [MIC(90)], > or = 16 mg/liter), and except for fluconazole (MIC(90), 8 mg/liter), the azole MICs were low: MIC(90)s were 0.5 mg/liter for itraconazole, 0.25 mg/liter for voriconazole, 0.25 mg/liter for posaconazole, and 0.125 mg/liter for isavuconazole. The echinocandins, caspofungin and anidulafungin, demonstrated no activity against Trichosporon species.

Cuban Cryptococcus isolates (n = 165) were tested in vitro against amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole, giving MIC90 values of 0.25, 8, 4, 0.25, 0.125, 0.016, and 0.016 microg/ml, respectively. Isavuconazole and posaconazole seem to be potentially active drugs for treating cryptococcal infections.