Bio-analytical liquid chromatographic-based method with a mixed mode online solid phase extraction for drug monitoring of fluconazole in human serum.

A simple, cost-effective and sensitive liquid chromatography-based bio-analytical method has been developed and validated for therapeutic drug monitoring of fluconazole (FLUC) in human serum. Integration of online mixed-mode solid-phase extraction (SPE) into the analytical system was the key for direct injection of untreated serum samples. A short protein-coated (PC) µBondapak CN silica column (PC-µB-CN-column) as a SPE tool and phosphate buffer saline (PBS) (pH 7.4) as an eluent were applied in the extraction step. PC-µB-CN-column operates in two different chromatographic modes. Using PBS, proteins were extracted from serum samples by size-exclusion liquid chromatography, while FLUC trapping was reversed-phase liquid chromatography dependent. FLUC was then eluted from the PC-µB-CN-column onto the quantification position using a mixture of acetonitrile-distilled deionized water (20:80, v/v) as an eluent and ODS analytical column. FLUC was separated at ambient temperature (22 ± 1 °C) and detected at 260 nm. The method was linear over the range of 200-10000 ng/mL. FLUC recovery in untreated serum samples ranged from 97.8 to 98.8% and showed good accuracy and precision. The reliability of the developed method was evaluated by studying the pharmacokinetic profile of FLUC in humans after an oral administration of a single 150 mg tablet.

Preparation of magnetic metal organic framework and development of solid phase extraction method for simultaneous determination of fluconazole and voriconazole in rat plasma samples by HPLC.

Fluconazole and voriconazole are the two broad-spectrum triazole antifungals. The present work described the fabrication method for the synthesis of the amino-modified magnetic metal-organic framework. This material was applied as a pre-sample treatment sorbent for the selective extraction of fluconazole and voriconazole in rat plasma samples. The material was fabricated by the chemical bonding approach method and was characterized by different parameters. The factors which affect the extraction efficiency of the sorbent material were also optimized in this study. Due to the optimization of solid-phase extraction conditions, the nonspecific interaction was reduced and the extraction recoveries of target drugs were increased in plasma samples. The extraction method was combined with the HPLC-UV method for the analysis. Excellent linearity (0.1-25 µg/mL), detections (0.02, 0.03 µg/mL) and quantification limits (0.04, 0.05 µg/mL) were resulted for fluconazole and voriconazole respectively. The maximum recoveries from spiked plasma samples of fluconazole and voriconazole were 86.8% and 78.6% and relative standard deviation were 0.9-2.8% and 2.2-3.6% respectively. Moreover, this sorbent material was used multiple times which was an improvement over single-use commercial sorbent materials. This validated method has practical potential for the simultaneous determination of these drugs in therapeutic drug monitoring studies as well as for routine pharmacokinetic evaluations.

Antifungal Activity of Capric Acid, Nystatin, and Fluconazole and Their In Vitro Interactions Against Candida Isolates from Neonatal Oral Thrush.

Due to the increasing resistance of various Candida species to azole drugs, particularly fluconazole, it would be of significant importance to look for alternative therapies. The aim of this study was to investigate the antifungal activity of capric acid and its in vitro interactions with nystatin and fluconazole against Candida isolates. A total of 40 Candida isolates (C. albicans, 36; C. kefyr, 2; C. tropicalis, 1; C. glabrata, 1) collected from the oral cavity of neonates with oropharyngeal candidiasis and a reference strain of C. albicans (ATCC 10231) were used in this study. Antifungal activity of capric acid and two comparator antifungal drugs, namely fluconazole and nystatin, was tested according to CLSI M27-A3/M60 method. The in vitro interaction between capric acid with fluconazole and nystatin was determined following a checkerboard method and results were interpreted using fractional inhibitory concentration index. Nystatin had the lowest minimum inhibitory concentrations (range, 0.125-8 µg/mL; geometric mean [GM], 0.6229 µg/mL) followed by fluconazole (range, 0.5-16 µg/mL; GM, 1.9011 µg/mL) and capric acid (range, 128-2,048 µg/mL; GM, 835.9756 µg/mL). When tested in combination, capric acid with fluconazole demonstrated synergistic, indifferent, and antagonistic interactions in 3 (7.317%), 24 (58.536%), and 14 (34.146%) cases, respectively. For combination of capric acid with nystatin, synergistic, indifferent, and antagonistic interactions were observed in 1 (2.439%), 19 (46.341%), and 21 (51.219%) cases, respectively. All cases of synergistic interactions were against resistant or susceptible dose-dependent isolates. Fluconazole, nystatin, and capric acid seem to be more effective when they are used alone compared with their combination. However, their combination might be effective on resistant isolates.

Isolation and structural identification of an impurity in fluconazole bulk drug substance.

Four impurities in fluconazole API sample obtained from a recently proposed synthetic process were detected by HPLC. One of the impurities was unknown having not been reported previously. This less polar unknown impurity was isolated from the crude sample of fluconazole bulk drug using semi-preparative HPLC. Structure of impurity was elucidated as 2-(2-(dimethylamino)-4-fluorophenyl)-1,3-di(3H-1,2,4-triazol-1-yl)propan-2-ol by using NMR spectroscopy(1H, 13C, 19F, 1H-1H, 1H-13C, HMBC and nOe) and mass spectrometry. The formation and synthesis of the impurity was discussed.

Unbound fraction of fluconazole and linezolid in human plasma as determined by ultrafiltration: Impact of membrane type.

Ultrafiltration is a rapid and convenient method to determine the free concentrations of drugs in plasma. Several ultrafiltration devices based on Eppendorf cups are commercially available, but are not validated for such use by the manufacturer. Plasma pH, temperature and relative centrifugal force as well as membrane type can influence the results. In the present work, we developed an ultrafiltration method in order to determine the free concentrations of linezolid or fluconazole, both neutral and moderately lipophilic antiinfective drugs for parenteral as well as oral administration, in plasma of patients. Whereas both substances behaved relatively insensitive in human plasma regarding variations in pH (7.0-8.5), temperature (5-37°C) or relative centrifugal force (1000-10.000xg), losses of linezolid were observed with the Nanosep Omega device due to adsorption onto the polyethersulfone membrane (unbound fraction 75% at 100mg/L and 45% at 0.1mg/L, respectively). No losses were observed with Vivacon which is equipped with a membrane of regenerated cellulose. With fluconazole no differences between Nanosep and Vivacon were observed. Applying standard conditions (pH 7.4/37°C/1000xg/20min), the mean unbound fraction of linezolid in pooled plasma from healthy volunteers was 81.5±2.8% using Vivacon, that of fluconazole was 87.9±3.5% using Nanosep or 89.4±3.3% using Vivacon. The unbound fraction of linezolid was 85.4±3.7% in plasma samples from surgical patients and 92.1±6.2% in ICU patients, respectively. The unbound fraction of fluconazole was 93.9±3.3% in plasma samples from ICU patients.

Simple means to alleviate sensitivity loss by trifluoroacetic acid (TFA) mobile phases in the hydrophilic interaction chromatography-electrospray tandem mass spectrometric (HILIC-ESI/MS/MS) bioanalysis of basic compounds.

Trifluoroacetic acid (TFA) is a commonly used additive in HPLC and LC-MS analysis of basic compounds. It is also routinely added to aqueous-organic mobile phases utilized in the hydrophilic interaction chromatography-electrospray tandem mass spectrometry (HILIC-ESI/MS/MS) technique used in our laboratories for bioanalysis. However, TFA is known to suppress the ESI signals of analytes due to its ability to form gas-phase ion pairs with positively-charged analyte ions. The most common method to overcome this problem involves the post-column addition of a mixture of propionic acid and isopropanol. However the post-column addition setup requires additional pumps and is not desirable for continuous analysis of large amounts of samples. In this paper we present a simple yet very effective means of minimizing the negative effect of TFA in bioanalysis by direct addition of 0.5% acetic acid or 1% propionic acid to mobile phases containing either 0.025 or 0.05% TFA. A factor of two- to five-fold signal enhancement was achieved for eight basic compounds studied. Furthermore, chromatography integrity was maintained even with the addition of acetic acid and propionic acid to existing TFA mobile phases. This method has been successfully applied to the HILIC-ESI/MS/MS high-throughput analysis of extracted biological samples to support pre-clinical and clinical studies.

A comparison between solid phase extraction and supercritical fluid extraction for the determination of fluconazole from animal feed.

The application of supercritical fluid extraction with carbon dioxide and modified carbon dioxide for the determination of fluconazole from an animal feed was studied. A fractional factorial design approach was used to examine the significant experimental variables for quantitative extraction of fluconazole. Gas chromatography with either flame ionisation or mass selective detection was used for quantitation of the extracts. The results indicated that modifier (methanol) had the greatest effect on the recovery of fluconazole from the animal feed.

Candida orthopsilosis fungemias in a Spanish tertiary care hospital: Incidence, epidemiology and antifungal susceptibility / Fungemias por Candida orthopsilosis en un hospital terciario español: incidencia, epidemiología y sensibilidad a antifúngicos

Background. Few studies exist on prevalence of fungemia by Candida orthopsilosis, with variable results. Aims. To study the incidence, epidemiology and antifungal susceptibility of C. orthopsilosis strains isolated from fungemias over two years at a tertiary hospital. Methods. Candidemia episodes between June 2007 and June 2009 in a university hospital (Puerta del Mar, Cádiz, Spain) were studied. The strains initially identified as Candida parapsilosis were genotypically screened for C. parapsilosis sensu stricto, C. orthopsilosis and Candida metapsilosis, and their antifungal susceptibility was evaluated. Results. In this period 52 cases of candidemia were documented. Of the 19 strains originally identified as C. parapsilosis, 13 were confirmed as C. parapsilosis sensu stricto and 6 as C. orthopsilosis. Of the 52 isolates, the most frequent species were Candida albicans (30.8%), C. parapsilosis sensu stricto (25%), C. orthopsilosis, Candida tropicalis and Candida glabrata in equal numbers (11.5%). C. orthopsilosis isolates were susceptible to amphotericin B, caspofungin, voriconazole and fluconazole, with no significant differences in MIC values with C. parapsilosis sensu stricto. The source of isolates of C. orthopsilosis were neonates (50%) and surgery (50%), and 100% were receiving parenteral nutrition; however C. parapsilosis sensu stricto was recovered primarily from patients over 50 years (69.2%) and 46.1% were receiving parenteral nutrition. Conclusions. These findings show that C. orthopsilosis should be considered as human pathogenic yeast and therefore its accurate identification is important. Despite our small sample size our study suggests that a displacement of some epidemiological characteristics previously attributed to C. parapsilosis to C. orthopsilosis may be possible (AU)

Antecedentes. Apenas se han publicado estudios sobre la prevalencia de los episodios de fungemia por Candida orthopsilosis, y sus resultados han sido variables. Objetivos. Examinar la incidencia, epidemiología y sensibilidad a antifúngicos de las cepas de C. orthopsilosis aisladas de fungemias en un periodo de 2 años en un hospital de asistencia terciaria. Métodos. Entre junio de 2007 y junio de 2009, en el Hospital Universitario Puerta del Mar (Cádiz, España) se estudiaron todos los episodios de fungemia. Las cepas identificadas inicialmente como Candida parapsilosis se genotipificaron para su clasificación como C. parapsilosis sensu stricto, C. orthopsilosis y Candida metapsilosis, y se testó su sensibilidad a los antifúngicos. Resultados. Durante este periodo, se documentaron 52 episodios de fungemia. De las 19 cepas identificadas originalmente como C. parapsilosis, 13 fueron C. parapsilosis sensu stricto, y 6 C. orthopsilosis. De los 52 aislamientos, las especies más frecuentes fueron Candida albicans (30,8%), C. parapsilosis sensu stricto (25%) y C. orthopsilosis (11,5%), y Candida tropicalis y Candida glabrata fueron aisladas en igual número. Todos los aislamientos de C. orthopsilosis fueron sensibles a anfotericina B, caspofungina, voriconazol y fluconazol, sin diferencias significativas en las concentraciones inhibitorias mínimas obtenidas con C. parapsilosis sensu stricto. Los aislamientos de C. orthopsilosis procedían de recién nacidos (50%) y de pacientes sometidos a cirugía (50%). El 100% de los pacientes recibía nutrición parenteral; sin embargo, el foco de C. parapsilosis sensu stricto procedía, ante todo, de pacientes de más de 50 años de edad (69,2%), y el 46,1% recibía nutrición parenteral. Conclusiones. Los resultados del presente estudio revelan que C. orthopsilosis debe considerarse una levadura patogénica para el ser humano y, por esta razón, es importante su identificación. A pesar del pequeño tamaño de la muestra, el presente estudio evidencia el desplazamiento a C. orthopsilosis de algunas características epidemiológicas atribuidas previamente a C. parapsilosis (AU)

Susceptibility tests of oropharyngeal Candida albicans from Egyptian patients to fluconazole determined by three methods

Candida albicans frequently cause oropharyngeal candidiasis in immunocompromised patients. As some of these isolates show resistance against azoles, the clinician is wary of initiating therapy with fluconazole (FZ) until a final susceptibility report is generated. We aimed to evaluate the efficacy of rapid flow cytometry (FCM) and disc diffusion (DD) methods in comparison to reference microdilution (MD) of Clinical and Laboratory Standards Institute (CLSI) method for FZ. Thirty seven Candida albicans isolates were tested by the three methods. By both MD and FCM, 26/37 (70.3%) were sensitive with minimal inhibitory concentration (MIC) ¡Ü 8¦Ìg/ml, 5/37 (13.5%) were susceptible dose dependant (S-DD) with MIC 16-32 ¦Ìg/ml and 6/37 (16.2%) were resistant with MIC ¡Ý64¦Ìg/ml. More than 92% of isolates susceptible to FZ by the MD were susceptible by the DD methods with good agreement (81.08%, P = 0.000). However, 4/5 isolates diagnosed as S-DD by MD were resistant by DD. Interestingly, the MIC by FCM at 4 h showed excellent agreement (95.59%, P = 0.000) to that obtained by MD method at 24 h. Overall, FCM antifungal susceptibility testing provided rapid, reproducible results that are valuable alternative to MD. The DD test is recommended as a simple and reliable screening test for the detection of susceptible Candida albicans isolates to FZ.

Application of capillary zone electrophoresis with off-line solid-phase extraction to in vitro metabolism studies of antifungals.

A simple and robust solid-phase extraction (SPE) procedure for the cleanup and sample preconcentration of antifungals (ketoconazole, clotrimazole, itraconazole, fluconazole, and voriconazole) and their metabolites after incubation with human liver microsomes, as well as a simplified capillary zone electrophoresis (CZE) method for their rapid analysis, have been developed to determine the stability of these compounds in in vitro samples. Three different sample pretreatment procedures using SPE with reversed-phase sorbents (100 mg C8, 100 mg C18, and 30 mg Oasis-HLB) were studied. The highest and most reproducible recoveries were obtained using a 30 mg Oasis-HLB sorbent and methanol containing 2% acetic acid as eluent. Enrichment by a factor of about four times was achieved by reconstituting the final SPE eluates to a small volume. For the CZE separation, good separations without interfering peaks due to the in vitro matrix were obtained with a simple running electrolyte using a fused-silica capillary. The best separation for all components originated by each tested drug after incubation with human liver microsomes (unmetabolized parent drug and its metabolites) was obtained using a 0.05 M phosphate running buffer (pH 2.2) without additives. The effect of the injection volume was also investigated in order to obtain the best sensitivity. Performance levels in terms of precision, linearity, limits of detection, and robustness were determined.

Membrane fluidity and lipid composition of fluconazole resistant and susceptible strains of Candida albicans isolated from diabetic patients / Fluidez e composição lipídica da membrana de cepas de Candida albicans resistentes e sensíveis ao fluconazol, isoladas de pacientes diabéticos

Ten clinical isolates of Candida albicans, five strains belonging to each of fluconazole resistant and susceptible groups isolated from diabetic patients, were studied for the membrane fluidity and lipid composition. Compared to fluconazole susceptible strains, fluconazole resistant ones exhibited enhanced membrane fluidity as measured by fluorescence polarization technique. The increased membrane fluidity was reflected in the decreased p-values exhibited by the resistant strains. On the other hand, susceptible isolates contained higher amount of ergosterol, almost twice as compared to resistant isolates which might have contributed to their lower membrane fluidity. However, no significant alteration was observed in the phospholipid and fatty acid composition of these isolates. Labeling experiments with fluorescamine dye revealed that the percentage of the exposed aminophospholipid, phosphatidylethanolamine was highest in the resistant strains as compared to the susceptible strains, indicating a possible overexpression of CDR1 and CDR2 genes in resistant strains. The results presented here suggest that the changes in the ergosterol content and overexpression of ABC transporter genes CDR1 and CDR2 could contributeto fluconazole resistance in C. albicans isolated from diabetic patients.

Dez isolados clínicos, sendo cinco resistentes e cinco sensíveis ao fluconazol, obtidos de pacientes diabéticos, foram estudados quanto à fluidez e composição química da membrana. Quando comparados aos isolados sensíveis ao fluconazol, os isolados resistentes apresentaram fluidez de membrana aumentada, conforme mensurado pela técnica de polarização fluorescente. A fluidez de membrana aumentada refletiu-se pelos valores mais baixos de p. Por outro lado, os isolados sensíveis continham quantidades mais elevadas de ergosterol, quase o dobro dos isolados resistentes, o que pode ter contribuído para a fluidez de membrana mais baixa. Entretanto, não se observou alteração significativa na composição fosfolipídica e de ácidos graxos nesses isolados. Experimentos de marcação com corante fluorescamina indicaram que a porcentagem de aminofosfolípides e fosfatidiletanolamina expostos foi mais elevada nos isolados resistentes do que nos sensíveis, indicando uma possível superexpressão dos genes CDR1 e CDR2 nos isolados resistentes. Os resultados aqui apresentados sugerem que alterações no teor de ergosterol e superexpressão dos genes ABC transportadores CDR1 e CDR2 podem contribuir na resistência ao fluconazol em isolados de C. albicans de pacientes diabéticos.

Sensibilidad a fluconazol de levaduras de interés clínico: nuevos puntos de corte / Susceptibility to fluconazole of clinical interest yeasts: new breakpoints

Introducción. Recientemente, Pfaller y colaboradores (Drug Resist Updat 2010; 13:180-95), han propuesto nuevos puntos de corte para determinar la sensibilidad in vitro a fluconazol de Candida albicans, C. parapsilosis y C. tropicalis. El objetivo de este trabajo ha sido establecer las variaciones de sensibilidad de estas especies al aplicar estos puntos de corte, en relación con los del Clinical and Laboratory Standards Institute (CLSI). Métodos. Analizamos 112 cepas de Candida: 49 C. albicans, 40 C. parapsilosis y 23 C. tropicalis. La sensibilidad a fluconazol se ensayó por el método Sensititre YeastOne. Los puntos de corte para categorizar la concentración mínima inhibitoria (CMI) fueron los del CLSI y los propuestos por Pfaller y colaboradores. Resultados. Según los criterios del CLSI, todas las cepas fueron sensibles a fluconazol. Las CMI50 y CMI90 fueron 0,5 mg/L y 2 mg/L para C. albicans y C. parapsilosis, 0,5 mg/L y 1 mg/L para C. tropicalis. Con los nuevos criterios, 109 (97%) cepas fueron sensibles. Solamente se apreciaron variaciones en C. albicans (6% sensibles dosis dependientes). Conclusiones. Al aplicar los puntos de corte recomendados por Pfaller y colaboradores, y los del EUCAST, el número de cepas sensibles a fluconazol disminuye en relación con los criterios del CLSI, especialmente de C. albicans(AU)

Introduction. Recently, Pfaller et al (Drug Resist Update 2010; 13:180-95), have proposed new breakpoints for determining the in vitro susceptibility to fluconazole of Candida albicans, C. parapsilosis and C. tropicalis. The aim of this study was to establish the variations in sensitivity of these species applying these breakpoints, in relation to those of the Clinical and Laboratory Standards Institute (CLSI). Methods. We analyzed 112 strains of Candida: 49 C. albicans, 40 C. parapsilosis and 23 C. tropicalis. Susceptibility to fluconazole was performed by the method Sensititre YeastOne. The breakpoints used to determine the minimum inhibitory concentration (MIC) were identified by CLSI and the ones proposed by Pfaller et al. Results. According to the CLSI criteria, all isolates were susceptible to fluconazole. MIC50 and MIC90 were 0.5 mg/L and 2 mg/L for C. albicans and C. parapsilosis, 0.5 mg/L and 1 mg/L for C. tropicalis. With the new criteria, 109 (97%) strains were susceptible. Variations were seen in C. albicans, with 3 strains (6%) susceptible dose-dependent. Conclusions. When applying the breakpoints recommended by Pfaller et al, and EUCAST, the number of fluconazole-susceptible strains decreased according to the CLSI criteria, especially C. albicans(AU)

Variación de la epidemiología de las fungemias y de la sensibilidad al fluconazol de los aislamientos de hemocultivos en los últimos 10 años en España: resultados del estudio FUNGEMYCA / Changes in the epidemiology of fungaemia and fluconazole susceptibility of blood isolates during the last 10 years in Spain: results from the FUNGEMYCA study

Antecedentes. Recientemente se ha observado un incremento de las fungemias causadas por especies diferentes de Candida albicans y una disminución de la sensibilidad de los microorganismos responsables al fluconazol. Objetivos. Evaluar la epidemiología y la sensibilidad al fluconazol de los casos de fungemia en España en 2009, comparando los resultados con los obtenidos entre los años 1997-1999 (Pemán J, et al. Eur J Clin Microbiol Infect Dis. 2005). Métodos. Estudio prospectivo multicéntrico con 44 centros participantes realizado desde enero de 2009 a febrero de 2010. Los aislamientos fúngicos procedentes de hemocultivo fueron recogidos en cada centro, donde se realizó el estudio de sensibilidad antifúngica mediante microdilución colorimétrica (Sensititre Yeast One). Resultados. Desde enero de 2009 a febrero de 2010 se recogieron 1.377 aislamientos en hemocultivos, correspondientes a 1.357 episodios de fungemia. Las fungemias se observaron principalmente en mayores de 64 años (46,7%) y el 8,6% en menores de 1 año. C. albicans (44,7%), Candida parapsilosis (29,1%), Candida glabrata (11,5%), Candida tropicalis (8,2%) y Candida krusei (1,9%) fueron las especies más frecuentes, pero su distribución no fue geográficamente homogénea. En los últimos 10 años la incidencia de C. albicans ha aumentado significativamente en Cataluña (39,1 vs. 54,7%, P=0,03) y reducido en la Comunidad Valenciana (49,1 vs. 34,6%, P=0,01). C. parapsilosis ha disminuido en Cataluña (29 vs. 12,4%, P=0,002) y Extremadura (58,3 vs. 20%, P=0,01). La sensibilidad a fluconazol fue similar en toda España pero en los aislamientos de C. albicans la resistencia fue diez veces superior en mayores de 64 años. Sin embargo, la tasa de resistencia (CMI > 32 mg/L) global ha disminuido con respecto a la obtenida hace 10 años (3,7 vs. 2,5% actual), sobre todo en C. albicans (3 vs. 1,6%). Conclusiones. En los últimos 10 años la distribución de las especies causantes de fungemia en España y la sensibilidad al fluconazol no han variado significativamente, aunque se observa una menor tasa de resistencia. La distribución de las especies varía según la unidad de hospitalización, hospital y Comunidad Autónoma(AU)

Background. Recent epidemiological surveillance studies have reported an increase in fungaemia caused by non-Candida albicans species, as well as a decrease in fluconazole susceptibility. Objectives. To evaluate changes in the epidemiology of fungaemia in Spain comparing data from a new surveillance epidemiological study conducted in 2009 with a previous study carried out from 1997 to 1999 (Pemán J, et al. Eur J Clin Microbiol Infect Dis. 2005). Methods. From January 2009 to February 2010, 44 Spanish hospitals participated in a prospective multicentre fungaemia surveillance study to ascertain whether there have been changes in the epidemiology and fluconazole susceptibility. Susceptibility was determined by the colorimetric method Sensititre Yeast One. Demographic and clinical data and the first isolate of each episode were gathered. Results. A total of 1,377 isolates from 1,357 fungaemia episodes were collected, 46.7% from patients older than 64years and 8.6% from children less than 1 year old. C. albicans (44.7%), Candida parapsilosis (29.1%), Candida glabrata (11.5%), Candida tropicalis (8.2%), and Candida krusei (1.9%) were the most frequent species isolated. Distribution varied with the geographical area. C. albicans incidence has increased significantly in the last 10years in Cataluña (39.1 vs. 54.7%, P =0.03) and decreased in the Valencian Community (49.1 vs. 34.6%, P =0.002) and Extremadura (58.3 vs. 20%, P =0.01). Susceptibility to fluconazole was similar for all geographical areas, although resistance in C. albicans was ten times greater for patients aged more than 64years. The overall rate of fluconazole resistance (MIC > 32 mg/L) has decreased with respect to that obtained 10years ago (3.7 vs. 2.5%) mainly in C. albicans (3 vs. 1.6%). Conclusions. In the last ten years, species distribution and fluconazole susceptibility have not significantly changed, although a lower rate of fluconazole resistance has been observed. Species distribution varies with hospital, hospitalization Unit and geographical area(AU)