SUSCEPTIBILITY TEST FOR FUNGI: CLINICAL AND LABORATORIAL CORRELATIONS IN MEDICAL MYCOLOGY.

During recent decades, antifungal susceptibility testing has become standardized and nowadays has the same role of the antibacterial susceptibility testing in microbiology laboratories. American and European standards have been developed, as well as equivalent commercial systems which are more appropriate for clinical laboratories. The detection of resistant strains by means of these systems has allowed the study and understanding of the molecular basis and the mechanisms of resistance of fungal species to antifungal agents. In addition, many studies on the correlation of in vitro results with the outcome of patients have been performed, reaching the conclusion that infections caused by resistant strains have worse outcome than those caused by susceptible fungal isolates. These studies have allowed the development of interpretative breakpoints for Candida spp. and Aspergillus spp., the most frequent agents of fungal infections in the world. In summary, antifungal susceptibility tests have become essential tools to guide the treatment of fungal diseases, to know the local and global disease epidemiology, and to identify resistance to antifungals.

Susceptibility eest for fungi: clinical and laboratorial correlations in medical mycology / Teste de suscetibilidade para fungos: correlações clínico-laboratoriais em Micologia médica

SUMMARYDuring recent decades, antifungal susceptibility testing has become standardized and nowadays has the same role of the antibacterial susceptibility testing in microbiology laboratories. American and European standards have been developed, as well as equivalent commercial systems which are more appropriate for clinical laboratories. The detection of resistant strains by means of these systems has allowed the study and understanding of the molecular basis and the mechanisms of resistance of fungal species to antifungal agents. In addition, many studies on the correlation of in vitro results with the outcome of patients have been performed, reaching the conclusion that infections caused by resistant strains have worse outcome than those caused by susceptible fungal isolates. These studies have allowed the development of interpretative breakpoints for Candida spp. and Aspergillus spp., the most frequent agents of fungal infections in the world. In summary, antifungal susceptibility tests have become essential tools to guide the treatment of fungal diseases, to know the local and global disease epidemiology, and to identify resistance to antifungals.

RESUMONas últimas décadas, os testes de suscetibilidade a antifúngicos foram padronizados e, atualmente, servem tal como os testes de suscetibilidade a antibacterianos em laboratórios de microbiologia. Métodos de referência norte americanos e europeus foram desenvolvidos, assim como os equivalentes sistemas comerciais, estes últimos mais apropriados a laboratórios clínicos. A detecção de cepas resistentes por meio de tais sistemas permitiu o estudo e a compreensão das bases moleculares dos mecanismos de resistência de espécies fúngicas a fármacos antifúngicos. Além disso, foram realizados muitos estudos sobre a correlação de resultados obtidos in vitro com o desfecho clínico de pacientes permitindo a conclusão de que infecções por cepas resistentes têm pior evolução em relação às causadas por cepas sensíveis. Os estudos permitiram o estabelecimento de pontos de corte interpretativos (interpretative breakpoints development) para Candida spp. e Aspergillus spp., os agentes etiológicos mais frequentes de infecções fúngicas em todo o mundo. Em resumo, os testes de suscetibilidade representam uma ferramenta essencial para a orientação do tratamento de doenças fúngicas, para o conhecimento da epidemiologia local e global, bem como para a identificação de resistência a antifúngicos.

Development and validation of a fast HPLC/photodiode array detection methodfor the measurement of voriconazole in human serum samples. A reference laboratory experience

The aim of this study was the development and validation of a fast and simple high performance liquid chromatography method for measuring voriconazole in human serum using ravuconazole as an external standard. The experience of the reference laboratory in therapeutic drug monitoring of voriconazoleis also reported. This method is based on the precipitation of proteins in human serum and detection by HPLC/UV. Chromatographic separation is achieved using an isocratic solvent delivery with detection at 255 nm and a run time of 7 min. The assay was validated according to international guidelines and was also applied to the analysis of 141 trough serum samples from patients treated with voriconazole. All validation parameters met the criteria set out in FDA guidelines for bioanalytical methods. A highinter patient and intrapatient variability was observed in clinical samples. This method is accurate enough to perform therapeutic drug monitoring in patients receiving voriconazole treatmen (AU)

El objetivo de este estudio fue el desarrollo y validación de un método rápido y sencillo mediante cromatografía líquida de alta eflcacia para la cuantiflcación de voriconazol en muestras de suero de origen humano usando ravuconazol como estándar de extracción. Además se detalla la experiencia de un centro de referencia en la monitorización de voriconazol. El método está basado en la precipitación de proteínas con acetonitrilo para su posterior procesamiento en un aparato de HPLC/UV. La separación cromatográflcase llevo a cabo usando flujo isocrático con longitud de onda de detección a 255 nm y un tiempo de análisis de 7 minutos. En ensayo se validó de acuerdo con las guías internacionales y se aplicó al análisis de 141muestras de pacientes tratados. Todos los parámetros de validación cumplían los criterios de la Guía de la FDA para la validación de métodos bioanalíticos. Se observó una gran variabilidad intra e interpaciente en las muestras clínicas evaluadas. El método es suflcientemente preciso y exacto para la monitorizacón de pacientes en tratamiento con voriconazol (AU)

Infección pulmonar recurrente por Aspergillus fumigatus con resistencia a múltiples triazoles en un paciente español con leucemia mieloide crónica / Breakthrough pulmonary Aspergillus fumigatus infection with multiple triazole resistance in a Spanish patient with chronic myeloid leukemia

Antecedentes. Un paciente sometido a un trasplante alogénico de progenitores hematopoyéticos se presentó con aspergilosis pulmonar crónica, asociada a enfermedad pulmonar injerto contra huésped, y fue tratado durante un período prolongado con diversos antimicóticos administrados como profilaxis, tratamiento de combinación y tratamiento de mantenimiento. El paciente experimentó una aspergilosis pulmonar invasiva recurrente debida a Aspergillus fumigatus tras el tratamiento antimicótico prolongado. Material y métodos. Se analizarán diversos aislamientos. Los primeros aislamientos eran sensibles in vitro a todos los azoles. No obstante, tras tratamiento prolongado con itraconazol y voriconazol, a partir de líquido de lavado broncopulmonar (LBA) cuando el paciente experimentó una infección invasiva, se cultivó un aislamiento de A. fumigatus resistente a múltiples azólicos y se observaron lesiones cavitarias. Resultados. El análisis de los mecanismos de resistencia que actuaron en la última cepa nos condujo a publicar un artículo sobre el primer aislamiento en España de una cepa de A. fumigatus resistente a un azol que albergaba la mutación L98H en combinación con una alteración de repeticiones en tándem (RT) en el gen CYP51A (TR-L98H). El tratamiento prolongado con un azol puede aumentar la selección de cepas con una disminución de la sensibilidad a estos fármacos. Sin embargo, en este caso, puesto que los aislamientos eran genéticamente diferentes, se sugirió que la resistencia no estuvo inducida durante el tratamiento prolongado con azólicos sino que el paciente adquirió este aislamiento resistente del entorno y fue seleccionado por el tratamiento. Conclusiones. Los hallazgos del presente estudio sugieren que en todos los tratamientos crónicos con antimicóticos, en particular con azólicos, puede ser necesaria la obtención de muestras repetidas, al igual que la realización de exámenes a intervalos regulares de la sensibilidad de las cepas aisladas, ya que pueden seleccionarse aislamientos resistentes(AU)

Background. An allogeneic hematopoietic cell transplantation (allo-HCT) patient presented with chronic pulmonary aspergillosis associated to pulmonary graft versus host disease (GVHD) and was treated for a long time with several antifungal agents that were administered as prophylaxis, combination therapies, and maintenance treatment. The patient suffered from a breakthrough invasive pulmonary aspergillosis due to Aspergillus fumigatus after long-term antifungal therapy. Material and methods. Several isolates were analyzed. First isolates were susceptible in vitro to all azole agents. However, after prolonged treatment with itraconazole and voriconazole a multiple azole resistant A. fumigatus isolate was cultured from bronchoalveolar lavage (BAL) when the patient was suffering from an invasive infection, and cavitary lesions were observed. Results. Analysis of the resistant mechanisms operating in the last strain led us to report the first isolation in Spain of an azole resistant A. fumigatus strain harboring the L98H mutation in combination with the tandem repeat (TR) alteration in CYP51A gene (TR-L98H). Long-term azole therapy may increase the risk of resistance selecting strains exhibiting reduced susceptibility to these compounds. However, since the isolates were genetically different the suggestion that could be made is that the resistance was not induced during the prolonged azole therapy but the patient might simply have acquired this resistant isolate from the environment, selected by the therapy. Conclusions. These findings suggest that in all long-term treatments with antifungal agents, especially with azoles, repeated sampling and regular susceptibility testing of strains isolated is necessary as resistant isolates could be selected(AU)

Breakthrough pulmonary Aspergillus fumigatus infection with multiple triazole resistance in a Spanish patient with chronic myeloid leukemia.

BACKGROUND: An allogeneic hematopoietic cell transplantation (allo-HCT) patient presented with chronic pulmonary aspergillosis associated to pulmonary graft versus host disease (GVHD) and was treated for a long time with several antifungal agents that were administered as prophylaxis, combination therapies, and maintenance treatment. The patient suffered from a breakthrough invasive pulmonary aspergillosis due to Aspergillus fumigatus after long-term antifungal therapy. MATERIAL AND METHODS: Several isolates were analyzed. First isolates were susceptible in vitro to all azole agents. However, after prolonged treatment with itraconazole and voriconazole a multiple azole resistant A. fumigatus isolate was cultured from bronchoalveolar lavage (BAL) when the patient was suffering from an invasive infection, and cavitary lesions were observed. RESULTS: Analysis of the resistant mechanisms operating in the last strain led us to report the first isolation in Spain of an azole resistant A. fumigatus strain harboring the L98H mutation in combination with the tandem repeat (TR) alteration in CYP51A gene (TR-L98H). Long-term azole therapy may increase the risk of resistance selecting strains exhibiting reduced susceptibility to these compounds. However, since the isolates were genetically different the suggestion that could be made is that the resistance was not induced during the prolonged azole therapy but the patient might simply have acquired this resistant isolate from the environment, selected by the therapy. CONCLUSIONS: These findings suggest that in all long-term treatments with antifungal agents, especially with azoles, repeated sampling and regular susceptibility testing of strains isolated is necessary as resistant isolates could be selected.

Development and validation of a fast HPLC/photodiode array detection method for the measurement of voriconazole in human serum samples. A reference laboratory experience.

The aim of this study was the development and validation of a fast and simple high performance liquid chromatography method for measuring voriconazole in human serum using ravuconazole as an external standard. The experience of the reference laboratory in therapeutic drug monitoring of voriconazole is also reported. This method is based on the precipitation of proteins in human serum and detection by HPLC/UV. Chromatographic separation is achieved using an isocratic solvent delivery with detection at 255 nm and a run time of 7 min. The assay was validated according to international guidelines and was also applied to the analysis of 141 trough serum samples from patients treated with voriconazole. All validation parameters met the criteria set out in FDA guidelines for bioanalytical methods. A high interpatient and intrapatient variability was observed in clinical samples. This method is accurate enough to perform therapeutic drug monitoring in patients receiving voriconazole treatment.

Comparison of dimethyl sulfoxide and water as solvents for echinocandin susceptibility testing by the EUCAST methodology.

Ninety-six strains of Candida, including 29 resistant and 67 susceptible isolates with mutations in the FKS1 and FKS2 genes were tested by the European Committee on Antibiotic Susceptibility Testing EDef 7.1 and 7.2 methodologies to determine the impact on the MIC when water was replaced with dimethyl sulfoxide (DMSO) as the solvent for caspofungin and micafungin. The MICs were significantly lower and the MIC ranges were narrower when DMSO was used as the solvent. The use of DMSO may help to better discriminate between susceptible and resistant populations.

Detection of invasive infection caused by Fusarium solani and non-Fusarium solani species using a duplex quantitative PCR-based assay in a murine model of fusariosis.

A duplex Real Time PCR (RT-PCR) assay for detecting DNA of members of the genus Fusarium has been developed and validated by using two mouse models of invasive infection. The duplex RT-PCR technique employed two specific molecular beacon probes targeting a highly conserved region of the fungal rDNA gene. This technique showed a detection limit of 10 fg DNA per µl of sample and a specificity of 100%. The sensitivity in a total of 48 samples from a murine model of Fusarium solani infection was 93.9% for lung tissues and 86.7% for serum samples. In comparison, the sensitivity in a total of 45 samples of a F. oxysporum murine model infection was 87% for lung tissues and 42.8% for serum samples. This molecular technique could be a reliable method for the quantification and the evaluation of the disease in animal models and for the clinical diagnosis of fusariosis.

Voriconazole serum levels measured by high-performance liquid chromatography: a monocentric study in treated patients.

In this study we present the results of a therapeutic drug monitoring retrospective analysis involving 14 patients with several underlying diseases who were receiving voriconazole for the treatment of fungal infections. A simple high performance liquid chromatography assay with ultraviolet detection was used in the drug monitoring. We report here that serum concentrations were highly variable and unpredictable in most patients. We also found that lack of response was more frequent in patients with levels persistently lower than 1 mg/l. The number of samples with voriconazole concentrations below 1 mg/l was significantly higher in patients who exhibited therapeutic failures (88% versus 27%; P < 0.001). In addition, the period of time in which voriconazole concentrations were maintained below 1 mg/l was slightly higher in patients in the failure group. We suggest that serum concentration should be individually quantified for patients receiving voriconazole therapy. Further prospective studies are needed to clarify the potential benefit of the individualization of treatment.

Role of Aspergillus lentulus 14-α sterol demethylase (Cyp51A) in azole drug susceptibility.

Recent studies have demonstrated that some morphologically atypical Aspergillus fumigatus strains are different species belonging to the section Fumigati. Aspergillus lentulus, one of these sibling species, is increasingly reported in patients under corticosteroid treatment. MICs of most antifungals in clinical use are elevated against A. lentulus, and it shows primary resistance to azole drugs. Two A. lentulus cytochrome P450 14-α sterol demethylases, encoded by A. lentulus cyp51A (Alcyp51A) and Alcyp51B genes, were identified. Targeted cyp51A gene knockout in A. lentulus showed that the intrinsic azole resistance of this species is cyp51A dependent. The Δcyp51A strain was morphologically indistinguishable from the A. lentulus wild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice. The heterologous expression of A. lentulus cyp51A was performed in an A. fumigatus cyp51A-deficient strain, confirming that Cyp51A is responsible for the differences in A. lentulus-azole drug interaction.

High-resolution melting analysis for identification of the Cryptococcus neoformans-Cryptococcus gattii complex.

We have developed a two-step method based on high-resolution melting (HRM) that reliably identifies species from the Cryptococcus species complex (Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii). Our results indicate that HRM can provide a fast protocol to identify and distinguish among the main Cryptococcus species.

Three-dimensional models of 14α-sterol demethylase (Cyp51A) from Aspergillus lentulus and Aspergillus fumigatus: an insight into differences in voriconazole interaction.

Aspergillus lentulus, an Aspergillus fumigatus sibling species, is increasingly reported in corticosteroid-treated patients. Its clinical significance is unknown, but the fact that A. lentulus shows reduced antifungal susceptibility, mainly to voriconazole, is of serious concern. Heterologous expression of cyp51A from A. fumigatus and A. lentulus was performed in Saccharomyces cerevisiae to assess differences in the interaction of Cyp51A with the azole drugs. The absence of endogenous ERG11 was efficiently complemented in S. cerevisiae by the expression of either Aspergillus cyp51A allele. There was a marked difference between azole minimum inhibitory concentration (MIC) values of the clones expressing each Aspergillus spp. cyp51A. Saccharomyces cerevisiae clones expressing A. lentulus alleles showed higher MICs to all of the azoles tested, supporting the hypothesis that the intrinsic azole resistance of A. lentulus could be associated with Cyp51A. Homology models of A. fumigatus and A. lentulus Cyp51A protein based on the crystal structure of Cyp51p from Mycobacterium tuberculosis in complex with fluconazole were almost identical owing to their mutual high sequence identity. Molecular dynamics (MD) was applied to both three-dimensional protein models to refine the homology modelling and to explore possible differences in the Cyp51A-voriconazole interaction. After 20ns of MD modelling, some critical differences were observed in the putative closed form adopted by the protein upon voriconazole binding. A closer study of the A. fumigatus and A. lentulus voriconazole putative binding site in Cyp51A suggested that some major differences in the protein's BC loop could differentially affect the lock-up of voriconazole, which in turn could correlate with their different azole susceptibility profiles.

Analysis of performance of a PCR-based assay to detect DNA of Aspergillus fumigatus in whole blood and serum: a comparative study with clinical samples.

The performance of a real-time PCR-based assay was retrospectively analyzed (according to European Organization for Research and Treatment of Cancer/Mycosis Study Group criteria) in the samples of patients with invasive aspergillosis. A total of 711 serial samples (356 whole-blood and 355 serum samples) from 38 adult patients were analyzed. The Aspergillus fumigatus PCR assay results were positive for 89 of 356 (25%) whole-blood samples and 90 of 355 (25.35%) serum samples. Positive PCR results were seen in 29 of 31 (93.5%) patients for which serum was analyzed and in 31 of 33 (93.9%) cases with whole-blood specimens. Both blood and serum samples were available in 26 cases, and significant differences were not observed in this subgroup of cases. The average number of threshold cycles (C(T)) for positive blood samples was 37.6, and the average C(T) for serum was 37.4. The DNA concentration ranged between 2 and 50 fg per µl of sample, with average DNA concentrations of 10.2 and 11.7 fg in positive blood and serum samples, respectively (P > 0.01). The performance of this PCR-based quantitative assay was similar for both serum and blood samples. We recommend serum samples as the most convenient hematological sample to use for Aspergillus DNA quantification when serial determinations are done.

Histoplasmosis and paracoccidioidomycosis in a non-endemic area: a review of cases and diagnosis.

BACKGROUND: Histoplasmosis and paracoccidioidomycosis (PCM) have increased in Spain in recent years, due firstly to the migration from endemic regions and secondly to travelers returning from these regions. In non-endemic areas, diagnosis of both diseases is hampered by the lack of experience, long silent periods, and the resemblance to other diseases such as tuberculosis and sarcoidosis. METHODS: A total of 39 cases of imported histoplasmosis and 6 cases of PCM diagnosed in the Spanish Mycology Reference Laboratory since 2006 were analyzed. Microbiological diagnosis was performed using classical methods and also a specific real-time polymerase chain reaction (RT-PCR) assay for each microorganism. RESULTS: We had 9 cases of probable histoplasmosis in travelers and 30 cases in immigrants, 29 of whom were defined as proven. Paracoccidioidomycosis (PCM) cases were either immigrants or people who had lived for a long period of time in endemic regions, all of whom were classified as proven cases. Cultures showed a good sensitivity in detecting Histoplasma capsulatum in immigrants with proven histoplasmosis (73%); however, growth was very slow. The fungus was never recovered in traveler patients. Paracoccidioides brasiliensis was isolated in a culture only in one case of the proven PCM. Serological methods were not very reliable in immunocompromised patients with histoplasmosis (40%). A PCR-based technique for histoplasmosis detected 55.5% of the cases in travelers (probable cases) and 89% of the cases in immigrants (proven). The PCR method for PCM detected 100% of the cases. CONCLUSIONS: These kinds of mycoses are increasingly frequent in non-endemic areas, and newer and faster techniques should be used to reach an early diagnosis. The RT-PCR techniques developed appear to be sensitive, specific, and fast and could be helpful to detect those mycoses. However, it is also essential that physicians perform differential diagnosis in individuals coming from endemic areas.

Recomendaciones sobre el diagnóstico de la enfermedad fúngica invasora de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC). Actualización 2010 / Spanish Society of Clinical Microbiology and Infectious Diseases (SEIMC) guidelines for the diagnosis of invasive fungal infections. 2010 update

Este texto incluye una actualización de las recomendaciones sobre el diagnóstico de la infección fúngica invasora de la Sociedad Española de Microbiología Clínica y Enfermedades Infecciosas (SEIMC) elaboradas en 2004 (Enferm Infecc Microbiol Clin. 2004, 22:32-9). En la actualización de 2010 se incluye una revisión exhaustiva de las novedades tecnológicas de los últimos años, así como los niveles de evidencia para recomendar cada una de las técnicas de diagnóstico. En primer lugar, se analizan los métodos convencionales, examen microscópico y cultivo, con sus limitaciones, lo que ha llevado a desarrollar métodos alternativos como la cuantificación de antígenos y de ADN fúngico. Las indicaciones de los métodos alternativos se analizan para las diferentes infecciones fúngicas, candidiasis, aspergilosis y micosis invasoras por otras especies. Por último, se incluye una breve descripción de los métodos de identificación molecular y se revisan las pruebas para realizar estudios de sensibilidad a los antifúngicos, los procedimientos de referencia, las técnicas comerciales y sus indicaciones (AU)

These guidelines are an update of recommendations for the diagnosis of invasive fungal infections by the Spanish Society of Clinical Microbiology and Infectious Diseases (SEIMC) published in 2004 (Enferm Infecc Microbiol Clin. 2004, 22:32-9). In this updated version of the guidelines, a comprehensive review of the most recent diagnostic innovations and levels of evidence to recommend those diagnostic procedures are included. We first analyse conventional diagnostic methods, microscopic examination and culture, underlining their limitations which have led to the development of alternative methods, such as fungal antigen and DNA quantification. Those alternative methods of diagnosis are analysed by fungal infection. We also briefly review the methods for molecular identification of fungal species and recommendations for carrying out susceptibility tests for antifungal drugs, including reference procedures, commercial techniques and their indications (AU)

[Spanish Society of Clinical Microbiology and Infectious Diseases (SEIMC) guidelines for the diagnosis of invasive fungal infections. 2010 update]. / Recomendaciones sobre el diagnóstico de la enfermedad fúngica invasora de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC). Actualización 2010.

These guidelines are an update of recommendations for the diagnosis of invasive fungal infections by the Spanish Society of Clinical Microbiology and Infectious Diseases (SEIMC) published in 2004 (Enferm Infecc Microbiol Clin. 2004, 22:32-9). In this updated version of the guidelines, a comprehensive review of the most recent diagnostic innovations and levels of evidence to recommend those diagnostic procedures are included. We first analyse conventional diagnostic methods, microscopic examination and culture, underlining their limitations which have led to the development of alternative methods, such as fungal antigen and DNA quantification. Those alternative methods of diagnosis are analysed by fungal infection. We also briefly review the methods for molecular identification of fungal species and recommendations for carrying out susceptibility tests for antifungal drugs, including reference procedures, commercial techniques and their indications.

EUCAST breakpoints for antifungals.

Susceptibility testing of fungi and development of interpretative breakpoints has become increasingly important due to the growing incidence of invasive fungal infections, the number and classes of antifungals, and the emerging reports of acquired resistance. The subcommittee on antifungal susceptibility testing of the European Committee on Antibiotic Susceptibility Testing (EUCAST) has developed standards for susceptibility testing of fermentative yeasts and molds as well as proposing breakpoints for fluconazole and voriconazole against Candida. The aim of this work is to describe the EUCAST process of setting breakpoints for antifungals. Five aspects are evaluated during the process of developing breakpoints: 1) the most common dosage used in each European country, 2) the definition of the wild-type population for each target microorganism at the species level and the determination of epidemiological cutoffs, 3) the drug's pharmacokinetics and 4) pharmacodynamics, including Monte Carlo simulations, and 5) the correlation of MICs with clinical outcome of patients treated with the compound. When insufficient data are available (e.g., due to lack of information on the clinical outcome of infections caused by isolates with an elevated MIC), epidemiological cutoff values, rather than breakpoints, are recommended until the necessary information becomes available.

Identification of pathogenic rare yeast species in clinical samples: comparison between phenotypical and molecular methods.

Species identification using both phenotypic and molecular methods and antifungal susceptibility tests was carried out with 60 uncommon clinical yeasts. Our data show that phenotypic methods were insufficient for correct identification (only 25%) and that most of the wrongly identified strains showed a resistant antifungal profile.

Comparison of the Vitek 2 antifungal susceptibility system with the clinical and laboratory standards institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) Broth Microdilution Reference Methods and with the Sensititre YeastOne and Etest techniques for in vitro detection of antifungal resistance in yeast isolates.

The commercial technique Vitek 2 system for antifungal susceptibility testing of yeast species was evaluated. A collection of 154 clinical yeast isolates, including amphotericin B- and azole-resistant organisms, was tested. Results were compared with those obtained by the reference procedures of both the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Two other commercial techniques approved for clinical use, the Etest and the Sensititre YeastOne, were included in the comparative exercise as well. The average essential agreement (EA) between the Vitek 2 system and the reference procedures was >95%, comparable with the average EAs observed between the reference procedures and the Sensititre YeastOne and Etest. The EA values were >97% for Candida spp. and stood at 92% for Cryptococcus neoformans. Intraclass correlation coefficients (ICC) between the commercial techniques and the reference procedures were statistically significant (P<0.01). Percentages of very major errors were 2.6% between Vitek 2 and the EUCAST technique and 1.6% between Vitek 2 and the CLSI technique. The Vitek 2 MIC results were available after 14 to 18 h of incubation for all Candida spp. (average time to reading, 15.5 h). The Vitek 2 system was shown to be a reliable technique to determine antifungal susceptibility testing of yeast species and a more rapid and easier alternative for clinical laboratories than the procedures developed by either the CLSI or EUCAST.