[Identification and in vitro antifungal susceptibility of vaginal Candida spp. isolates to fluconazole, clotrimazole and nystatin]. / Identificación y sensibilidad antifúngica in vitro de Candida spp. de origen vaginal a fluconazol, clotrimazol y nistatina.

OBJECTIVE: The aim of this study was to identify and determine the in vitro antifungal susceptibility testing to clotrimazole, fluconazole, and nystatin of 145 clinical isolates of Candida spp. METHODS: M27-A3 microdilution method was used to determine minimal inhibitory concentrations (MIC) and partial MICs (MIC50 and MIC90) of drugs. A total of 145 isolates were studied, 126 were C. albicans, 16 C. glabrata, 2 C. parapsilosis y 1 C. tropicalis. RESULTS: MIC50 and MIC90 for FLZ against C. albicans were 0.25 mg/L and 1 mg/L respectively and for C. glabrata was achieved at 8 mg/L and 16 mg/L for fluconazole. Five isolates of C. albicans and one isolate of C. tropicalis were in vitro resistant to fluconazole (M27-S4). In C. albicans MIC50 and MIC90 for clotrimazole were of 0.03 mg/L and 0.06 mg/L, respectively. These values for C. glabrata were 0.25 mg/L and 1 mg/L, respectively. Five C. glabrata and 1 C. tropicalis were in vitro resistant to clotrimazole. MIC50 and MIC90 of nystatin were of 1 mg/L and 2 mg/L, respectively for C. albicans and C. glabrata. CONCLUSIONS: In this study, C. albicans is the most frequently isolated yeast, followed by C. glabrata. The antifungals tested were found to be in vitro active for the isolates, except for 6 isolates for fluconazole and 6 to clotrimazole.

La escala modificada de Bruselas como predictor de mortalidad en la Unidad de Terapia Intensiva / The modified Brussels scale as a predictor of mortality in the Intensive Care Unit

Objetivo Comparar la discriminación y calibración de la escala modificada de Bruselas con el sistema Simplified Acute Physiology Score version 3 (SAPS-3) como predictor de mortalidad. Diseño Cohorte prospectiva. Ámbito Unidad de Cuidados Intensivos Respiratorios (UCIR) del Hospital General de México. Pacientes De 285 ingresos en la UCIR en 2012, se incluyeron 251 pacientes, con una edad media de 48,4±17,1 años; de ellos, 132 fueron hombres (52,8%).Intervenciones Ninguna. Variables de interés Demográficas, SAPS-3 y escala modificada de Bruselas del primer día de estancia en UCIR. Resultados El valor de la escala de Bruselas modificada del día 1 fue 4,7±3,8, y de SAPS-3, 54,7±17,8. El área ROC para la escala de Bruselas modificada del día 1 fue 0,837±0,025 (IC 95% 0,787 a 0,887), y para SAPS-3, de 0,813±0,027 (IC 95% 0,761 a 0,865), con prueba de bondad de ajuste de Hosmer-Lemeshow de 5.885 (p=0,660) y 4.026 (p=0,855), respectivamente. Conclusiones La escala modificada de Bruselas en el día 1 tiene una discriminación y calibración excelentes para predecir la mortalidad de los pacientes en la UCIR, comparables a las del modelo SAPS-3 (AU)

Purpose To compare discrimination and calibration of the modified Brussels score with the Simplified Acute Physiology Score version 3 (SAPS-3) in predicting mortality. Design A prospective cohort study was carried out. Setting The Respiratory Intensive Care Unit (RICU) of Mexico General Hospital. Patients A total of 251 patients out of 285 admissions to the RICU in 2012 were included. The mean age was 48.4±17.1 years, and 132 of the patients were men (52.8%). Interventions None. Variables of interest Demographic data, SAPS-3 score upon admission and the modified Brussels score on the day 1 of stay in the RICU. Results On day 1, the modified Brussels and SAPS-3 scores were 4.7±3.8 and 54.7±17.8, respectively. Areas under the ROC curve for the modified Brussels score on day 1 and the SAPS-3 were 0.837±0.025 (95% CI 0.787-0.887) and 0.813±0.027 (95% CI 0.761-0.865), respectively. Hosmer-Lemeshow goodness-of-fit values were 5,885 (P=.660) and 4,026 (P=.855), respectively. Conclusions The modified Brussels score on day 1 offers excellent discrimination and calibration in predicting mortality in the RICU, comparable to that of the SAPS-3 (AU)

[The modified Brussels scale as a predictor of mortality in the Intensive Care Unit]. / La escala modificada de Bruselas como predictor de mortalidad en la Unidad de Terapia Intensiva.

PURPOSE: To compare discrimination and calibration of the modified Brussels score with the Simplified Acute Physiology Score version 3 (SAPS-3) in predicting mortality. DESIGN: A prospective cohort study was carried out. SETTING: The Respiratory Intensive Care Unit (RICU) of Mexico General Hospital. PATIENTS: A total of 251 patients out of 285 admissions to the RICU in 2012 were included. The mean age was 48.4±17.1 years, and 132 of the patients were men (52.8%). INTERVENTIONS: None. VARIABLES OF INTEREST: Demographic data, SAPS-3 score upon admission and the modified Brussels score on the day 1 of stay in the RICU. RESULTS: On day 1, the modified Brussels and SAPS-3 scores were 4.7 ± 3.8 and 54.7 ± 17.8, respectively. Areas under the ROC curve for the modified Brussels score on day 1 and the SAPS-3 were 0.837 ± 0.025 (95% CI 0.787-0.887) and 0.813 ± 0.027 (95% CI 0.761-0.865), respectively. Hosmer-Lemeshow goodness-of-fit values were 5,885 (P=.660) and 4,026 (P=.855), respectively. CONCLUSIONS: The modified Brussels score on day 1 offers excellent discrimination and calibration in predicting mortality in the RICU, comparable to that of the SAPS-3.

La escala modificada de bruselas como predictor de mortalidad en la unidad de terapia intensiva / The modified brussels scaleasa predictor of mortalityin the intensive care unit

Objetivo. Comparar la discriminación y calibración de la escala modificada de Bruselas con el sistema Simplified Acute Physiology Score version 3 (SAPS-3) como predictor de mortalidad. Diseño Cohorte prospectiva. Ámbito Unidad de Cuidados Intensivos Respiratorios (UCIR) del Hospital General de México. Pacientes De 285 ingresos en la UCIR en 2012, se incluyeron 251 pacientes, con una edad media de 48,4±17,1 años; de ellos, 132 fueron hombres (52,8%).I ntervenciones Ninguna.Variables de interésDemográficas, SAPS-3 y escala modificada de Bruselas del primer día de estancia en UCIR. Resultados. El valor de la escala de Bruselas modificada del día 1 fue 4,7±3,8, y de SAPS-3, 54,7±17,8. El área ROC para la escala de Bruselas modificada del día 1 fue 0,837±0,025 (IC 95% 0,787 a 0,887), y para SAPS-3, de 0,813±0,027 (IC 95% 0,761 a 0,865), con prueba de bondad de ajuste de Hosmer-Lemeshow de 5.885 (p=0,660) y 4.026 (p=0,855), respectivamente. Conclusiones. La escala modificada de Bruselas en el día 1 tiene una discriminación y calibración excelentes para predecir la mortalidad de los pacientes en la UCIR, comparables a las del modelo SAPS-3.

Sertaconazole antifungal profile determined by a microdilution method versus nine topical substances against dermatophyte fungi.

Antifungal activity and in vitro inhibition time for sertaconazole (STZ) and 9 other topical drugs, namely amorolfine, bifonazole, clotrimazole, econazole, ketoconazole, miconazole, oxiconazole, terbinafine, and tioconazole were determined against 124 clinical isolates of dermatophyte (12 species) fungi by the microdilution method in a liquid medium and the measurement of optical density. STZ's antifungal activity was not always affected by the tested dermatophyte genus, as was the case with the remaining antifungals. In vitro antifungal activity was at the same level for all the studied azole derivatives, but, in terms of partial inhibitory concentrations, STZ starts its in vitro inhibitory activity in a shorter time than the other tested substances, particularly in those incubation periods when the growth of the dermatophyte fungi was more developed.

Biofilm formation by five species of Candida on three clinical materials.

Most recalcitrant infections are associated with colonization and microbial biofilm development. These biofilms are difficult to eliminate by the immune response mechanisms and the current antimicrobial. Fungi can form biofilms on biomaterials commonly used in clinical practice (intravascular catheters, dentures, heart valves, implanted devices, contact lenses and other devices) and are associated with infections. A variety of in vitro models using different substrates/devices have been described. These models have been used to investigate the effect of different variables, including flow, growth time, nutrients and physiological conditions on fungal biofilm formation, morphology and architecture. The purpose of our study is to analyze biofilm formation capacity by 84 strains of Candida spp. (23C. albicans, 23C. parapsilosis, 16C. tropicalis, 17C. glabrata and 5C. krusei) on three materials used in medical devices and its quantification using a method based on viable cell count. Under the conditions of our study, all assayed Candida strains have been able to form biofilms. All species showed greater biofilm formation capacity on Teflon™, with the exception of C. glabrata which displayed higher biofilm formation capacity on PVC. Biofilm formation by Candida spp. varies depending on the type of material on which it grows and on the species and strain of Candida. The method we propose could be of great use to deepen scientific knowledge on this subject of remarkable clinical significance, considering the absence of standard biofilm formation and quantification techniques on the catheters and the level of difficulty associated to those available.

Antifungal activity of posaconazole against Candida spp. and non-Candida clinical yeasts isolates.

The in vitro antifungal activity of posaconazole was tested against 315 yeast clinical isolates and 11 ATCC reference strains by means an agar diffusion method (Neosensitabs, Rosco,Denmark) based in CLSI M44-A2 document. Posaconazole activity was excellent against Cryptococcus and Rhodotorula species studied and showed very good activity against most species of Candida tested. A total of 13 clinical isolates (4.1%) were resistant: Candida albicans (n=5), Candida glabrata (n=5), Candida tropicalis (n=1), Geotrichum australiensis (n=1) and Geotrichum capitatum (n=1). Our results suggest posaconazole is an effective antifungal agent against the most clinically important yeasts species (92.7% of susceptibility). Agar diffusion method provides good conditions for the posaconazole susceptibility study in the routine laboratory.

Activity of caspofungin and voriconazole against clinical isolates of Candida and other medically important yeasts by the CLSI M-44A disk diffusion method with Neo-Sensitabs tablets.

In vitro activity of caspofungin and voriconazole against 184 clinical isolates of Candida and other medically important yeasts in comparison with that of fluconazole, ketoconazole, itraconazole and amphotericin B was determined by using a disk diffusion method (Neo-Sensitabs) standardized according to the recommendations of the CLSI documents M44-A and M44-S1 (same medium: Mueller-Hinton plus methylene blue; inoculum and minimal inhibitory concentration/zone breakpoints). Seventy-two percent of clinical isolates were susceptible to caspofungin, 23.6% showed an intermediate susceptibility (most of them were Candida parapsilosis) and 4.3% were resistant (values for Candida spp. were 71.2, 23.8 and 5%, respectively). For voriconazole, 96.7% of clinical isolates were susceptible and 3.3% were resistant (Candida spp.: 96 and 3.8%, respectively). Both caspofungin and voriconazole showed high activity against a wide variety of clinically important yeasts.

In vitro activity of voriconazole against dermatophytes, Scopulariopsis brevicaulis and other opportunistic fungi as agents of onychomycosis.

Using a reference microdilution method, we studied the antifungal susceptibility to voriconazole and fluconazole of 304 clinical isolates from four species of onychomycosis-causing dermatophytes, 196 isolates of dermatophytes not related to nail infection as well as Scopulariopsis brevicaulis, Fusarium spp. and Scytalidium dimidiatum. Results showed a high antifungal activity of voriconazole against dermatophytes (geometric mean minimal inhibitory concentration (MIC)=1.14 microg/mL; MIC for 50% of the organisms (MIC(50))=0.062 miccrog/mL; MIC for 90% of the organisms (MIC(90))=0.25 microg/mL). For S. brevicaulis, the in vitro activity of voriconazole was considerably lower (geometric mean MIC=8.52 microg/mL; MIC(50) and MIC(90)=16 microg/mL). Although voriconazole is not among the drugs recommended for the management of onychomycosis, it can be a useful alternative for recalcitrant infections.

Comparison of tablet and disk diffusion methods for fluconazole and voriconazole in vitro activity testing against clinical yeast isolates.

We have compared a commercially available tablet diffusion method for the in vitro antifungal susceptibility testing of fluconazole (FCZ) and voriconazole (VCZ) with the disk diffusion method M44 (CLSI) with 282 clinical yeast isolates. The superior stability of antifungal agents in tablets can explain the differences for each category of susceptibility by both methods.Neo-Sensitabs tablets antifungal susceptibility testing showed an excellent correlation (0.98 for FCZ and 0.98 for VCZ at 24h and 0.96 for FCZ and 0.94 for VCZ at 48 h ), a reduced percentage of disagreements (4.6% and 8.2% for FCZ at 24h and 48 h respectively; 1.1% and 2.1% for VCZ at 24h and 48 h respectively) and the absence of statistically significant difference in comparison with the reference protocol for performing antifungal susceptibility testing with the agar diffusion method.

Antifungal agents: mode of action in yeast cells.

Different kinds of mycoses, especially invasive, have become an important public health problem as their incidence has increased dramatically in the last decades in relation to AIDS, hematological malignancies, transplant recipients and other immunosuppressed individuals. Management of fungal infections is markedly limited by problems of drug safety, resistance and effectiveness profile. Current therapy for invasive mycoses uses a relatively reduced number of antifungal drugs, such as amphotericin B, fluconazole and itraconazole. Other new antifungal agents from old and new chemical families, like voriconazole, posaconazole, ravuconazole, caspofungin and micafungin, have been introduced into the armamentarium for fungal infections management. This review is focused on the mode of action of those antifungal drugs used against pathogenic yeasts. The interaction of amphotericin B with ergosterol and other membrane sterols results in the production of aqueous pores of drug and the ergosterol biosynthetic pathway is the target of the allylamines, phenylmorpholines and azole antifungal agents. The main molecular target of azole antifungals is the cytochrome P-450 protein Erg11p/Cyp51p. Echinocandins, a new class of antifungal drugs, are fungal secondary metabolites that act against beta-1-3-D-glucan synthesis. The phenylmorpholines, of which amorolfine is the sole representative in human therapy, affect two targets in the ergosterol pathway: Erg24p (delta 14 reductase) and Erg2p (delta 8-delta 7 isomerase). The sordarins group are protein synthesis inhibitors that work by blocking the function of fungal translation elongation factor 2. Other protein inhibitors are zofimarin, BE31045, SCH57504, xylarin, hypoxysordarin and GR135402. In order to overcome the problems derived from the exploitation of azole drugs, macrolides and echinocandins, novel targets were explored. Proposed antifungal drugs have been developed against potential targets like the N-myristylation of fungal proteins, with inhibitors like myristate and histidine analogues or myristoylpeptide derivatives, aminobenzothiazoles, quinolines and benzofurans. Polymerization of cell wall carbohydrates from uridine di-phospho sugars is another potential target.

In vitro antifungal susceptibility testing of filamentous fungi with Sensititre Yeast One.

Sensititre is a colorimetric microdilution method for in vitro antifungal susceptibility testing based on the M27-A document (National Committee for Clinical Laboratory Standards) for yeasts. Difference between both methods is the presence of Alamar-blue and RPMI 1640 (glucose 2%) as culture medium. Antifungal susceptibility to amphotericin B, fluconazole, itraconazole, ketoconazole and flucytosine, 100 opportunistic filamentous fungi (Aspergillus spp., Fusarium spp., Scedosporium spp.) obtained from pathological samples was determined by the Sensititre method. Induction to conidium and sporangiospore formation at 35 degrees C was used to get inoculum and plates were covered by 1 ml of saline and suspensions were made by gently probing by a sterile loop. Optical densities of the conidial suspensions were adjusted to 80-82% transmittance for Aspergillus spp. and 68-70% for the rest of strains tested. Final inoculum concentration size was 0.4 x 10(4)-5 x 10(4) CFU ml(-1). Readings were made at 72 h of incubation at 35 degrees C; amphotericin B and itraconazole was active against Aspergillus fumigatus with CMI90 1 and 0.5 microg ml(-1), respectively, opposite to Scedosporium prolificans and Scedosporium apiospermum. As it was expected, a CMI90 of 256 microg ml(-1) for fluconazole and CMI90 for flucytosine amounting to 64 g ml(-1) were obtained. Sensititre Yeast One is a useful method and an alternative to reference methods to determine antifungal susceptibility of filamentous fungi for clinical laboratory routine. Correlation with microdilution results is studied. New triazole derivatives should be included as soon as their clinical use will be feasible.

[Is amphotericin B active against dermatophytes and Scopulariopsis brevicaulis?]. / Es activa la amfotericina B frente a hongos dermatófitos y Scopulariopsis brevicaulis?

The in vitro antifungal activity of amphotericin B was compared with that of griseofulvin, ketoconazole, clotrimazole and terbinafine in 193 clinical isolates of dermatophytes and Scopulariopsis brevicaulis. An agar diffusion method was used (NeoSensitabs) to categorize the susceptibility of the isolates as susceptible, intermediate or resistant to the antifungal agents. Using this method and following a standardized protocol adapted to the growth conditions of the dermatophytes and the opportunistic mold S. brevicaulis (inoculum size, temperature and time period of incubation), it was found that the in vitro susceptibility rates were 72%, 94.3%, 81.9%, 72% and 86% for amphotericin B, terbinafine, griseofulvin, ketoconazole and clotrimazole, respectively. Resistance percentages were 12.4%, 3.6%, 18.1%, 10.4% and 4.1% for the same antifungal agents. Amphotericin B showed no antifungal activity against S. brevicaulis; its activity against dermatophytes was similar to that of ketoconazole, and lower than that for clotrimazole and terbinafine.

In vitro antifungal activity of sertaconazole compared with nine other drugs against 250 clinical isolates of dermatophytes and Scopulariopsis brevicaulis.

We have tested 250 strains belonging to 15 species of clinically important dermatophytes and Scopulariopsis against ten antifungal drugs using an agar diffusion method (NeoSensitabstrade mark, Rosco, Taastrup, Denmark). Some of the experimental factors were adapted to dermatophyte development, such as temperature (28 vs. 35 degrees C) and time of incubation (2-5 days vs. 21-74 h). The antifungals used are itraconazole, ketoconazole, miconazole, clotrimazole, sertaconazole, terbinafine, tioconazole, fluconazole, isoconazole and econazole. Except for fluconazole, all the drugs tested have shown to be highly effective, especially sertaconazole and terbinafine. Percentages of susceptibility ranged between 94% for terbinafine, 87.6% for sertaconazole and 86.4% clotrimazole; 81.6% econazole; 42.8% fluconazole; 57.2% isoconazole; 78.4% itraconazole; 74.4% ketoconazole; 73.3% miconazole, and 85.2% for tioconazole. Percentages of resistance were similar between sertaconazole and terbinafine (4%) but in contrast to the 48% obtained for fluconazole.

Antifungal activity of amphotericin B and itraconazole against filamentous fungi: comparison of the Sensititre Yeast One and NCCLS M38--a reference methods.

The susceptibilities of 81 clinical isolates of Aspergillus spp., Fusarium spp., and Scedosporium spp., to amphotericin B and itraconazole were determined by the colorimetric microdilution method Sensititre and the reference microdilution method of NCCLS standard M38-A for filamentous fungi. No major discrepancies were found and agreement ranged between 86.4% to 84% and 69.1% to 86.4% for amphotericin B and itraconazole respectively at 48 h and 72 h of incubation by using the recommended endpoints. Within two two-fold dilutions, high levels of agreement were found in general for amphotericin B at 48 or 72 h (86.4 to 87.7%) and itraconazole (91.4 to 93.8%). Relatively better agreement was found for itraconazole at 72 h of incubation and 48 for amphotericin B.

Comparative evaluation of four commercial tests for presumptive identification of Candida albicans.

Four commercially available tests (Albicans ID2, Chromalbicans Agar, CHROMagar Candida, and BactiCard Candida) and the germ tube (GT) test for presumptive identification of Candida albicans were evaluated using clinical isolates of C. albicans (n=89) and of non-albicans yeasts (n=107). Sensitivities and specificities of all tests regarding the identification of C. albicans were greater than 92%, except for Chromalbicans Agar plates (88.7% after 48 h) and their specificity was 86%. Overall, the four commercial systems were easy to use and are good systems for the routine identification of C. albicans.

Performance of Bacticard Candida compared with the germ tube test for the presumptive identification of Candida albicans.

Bacticard Candida was compared with the germ tube test for the rapid, presumptive identification of Candida albicans. This test kit detects the enzymatic activities l-proline aminopeptidase and beta-galactosaminidase in yeast colonies grown on culture media. Candida albicans produces both enzymes whereas other yeasts produce only one or neither of the enzymes. We evaluated 536 isolates including eight genera and 33 species of medically important yeasts, including 228 C. albicans and 36 C. dubliniensis. Both tests did not discriminate between C. albicans and C. dubliniensis isolates. The sensitivity and specificity for the Bacticard Candida test were 97.8 and 96.5%, respectively. Bacticard Candida and germ tube tests detected 246 (93.2%), and 256 (97%) C. albicans plus C. dubliniensis isolates. There were eight false-positive results with BactiCard Candida kit and four false-positive results with the germ tube test. Positive and negative predictive values for Bacticard Candida enzymatic test were 95.3 and 98.4%, respectively, while 97.4 and 98.1% for the germ tube test, its specificity being 98.1% and efficiency 97% (97.7% for germ tube). We have observed slightly lower values of sensitivity and specificity than those reported by others using the BactiCard test kit. Bacticard Candida provides a rapid and accurate alternative to the germ tube test for the presumptive identification of C. albicans.