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All Malassezia species are lipophilic; thus, modifications are required in susceptibility testing methods to ensure their growth. Antifungal susceptibility of Malassezia species using agar and broth dilution methods has been studied. Currently, few tests using disc diffusion methods are being performed. The aim was to evaluate the in vitro susceptibility of Malassezia yeast against antifungal agents using broth microdilution and disc diffusion methods, then to compare both methodologies. Fifty Malassezia isolates were studied. Microdilution method was performed as described in reference document and agar diffusion test was performed using antifungal tablets and discs. To support growth, culture media were supplemented. To correlate methods, linear regression analysis and categorical agreement was determined. The strongest linear association was observed for fluconazole and miconazole. The highest agreement between both methods was observed for itraconazole and voriconazole and the lowest for amphotericin B and fluconazole. Although modifications made to disc diffusion method allowed to obtain susceptibility data for Malassezia yeast, variables cannot be associated through a linear correlation model, indicating that inhibition zone values cannot predict MIC value. According to the results, disc diffusion assay may not represent an alternative to determine antifungal susceptibility of Malassezia yeast., (© 2016 Blackwell Verlag GmbH.)

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.

Current therapy for mycoses is limited to the use of a relative reduced number of antifungal drugs. Although amphotericin B still remains considered as the "gold standard" for treatment, acute and chronic toxicity, such as impairment of renal function, limits its use and enhances the investigation and clinical use other chemical families of antifungal drugs. One of these chemical class of active drugs are azole derivatives, discovered in 70s and introduced in clinical practice in 80s. Being the most prolific antifungal class, investigation about more molecules, with a safer and better pharmacological profile, active against a wide spectrum of fungi, with a wide range of administration routes gives us some azole representatives.

We studied the in vitro antifungal activity profile of amorolfine (AMR), bifonazole (BFZ), clotrimazole (CLZ), econazole (ECZ), fluconazole (FNZ), itraconazole (ITZ), ketoconazole (KTZ), miconazole (MNZ), oxiconazole (OXZ), tioconazole (TCZ) and terbinafine (TRB) against 26 clinical isolates of Scopulariopsis brevicaulis from patients with onychomycosis by means of an standardized microdilution method. Although this opportunistic filamentous fungi was reported as resistant to several broad-spectrum antifungals agents, obtained data shows a better fungistatic in vitro activity of AMR, OXZ and TRB (0.08, 0.3, and 0.35 mg/L, respectively) in comparison to that of CLZ (0.47 mg/L), ECZ (1.48 mg/L), MNZ (1.56 mg/L, BFZ (2.8 mg/L), TCZ (3.33 mg/L), KTZ (3.73 mg/L). FNZ (178.47 mg/L) and ITZ (4.7 mg/L) showed a reduced in vitro antifungal activity against S. brevicaulis. Obtained MICs show the low in vitro antifungal susceptibility of S. brevicaulis to topical drugs for onychomycosis management, with exceptions (AMR, OZX and TRB).

Background: Dermatophytes are a group of keratinophilic fungi able to produce dermatophytosis or tinea infections. In Chile, Trichophyton rubrum and Trichophyton mentagrophytes are the ones most commonly isolated in adults, while Microsporum canis is found among children. Treatment of these infections is usually with topical or oral antifungals, such as griseofulvin or azole derivatives (clotrimazole, itraconazole, fluconazole), allylamines (terbinafine) or new drugs that are available., Aims: Evaluation of the in vitro susceptibility of dermatophytes to five antifungal agents and the comparison of the susceptibility pattern with that of previous years., Methods: Sixty-two clinical isolates of dermatophyte fungi were studied (March-June 2010). The CLSI M38-A2 micromethod was used., Results: Fluconazole MIC values for T. rubrum and T. mentagrophytes varied between 0.25 and 1 μg/ml; MIC range to clotrimazole, terbinafine and itraconazole was 0.03-0.06 μg/ml, and MIC values for griseofulvin were 0.015-0.03 μg/ml. No statistically significant differences were found between susceptibility patterns, except for fluconazole., Conclusions: Fluconazole was less active in comparison with other drugs tested (0.25-1 μg/ml). None of the isolates were resistant to any of the drugs, and no changes in the susceptibility pattern were observed when comparing the results with data previously reported concerning dermatophytes in Chile., (Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.)

Background: Malassezia pachydermatis is part of the skin microbiota of dogs and cats. M. pachydermatis has been associated with external otitis and seborrhoeic dermatitis, reported more often in dogs than in cats. When the physical, chemical or immunological mechanisms of the skin are altered, M. pachydermatis could act as a pathogen. Thus, several virulence factors, such as the ability to produce esterase, lipase, lipoxygenase, protease, chondroitin sulphatase, and hyaluronidase, have been studied., Aims: In the present study, we aim to identify the phospholipase activity measured at pH 6.3, and the proteinase activity measured at pH 6.3 and pH 6.8 (pH from ears of dogs with external otitis) of M. pachydermatis strains isolated from dogs with and without external otitis., Methods: The phospholipase activity was measured using a semi-quantitative method with egg yolk, and the proteinase activity with a semi-quantitative method using bovine serum albumin agar. The study was performed on 96 isolates of M. pachydermatis, 43 isolated from dogs without clinical symptoms of otitis, and 52 isolated from dogs with otitis., Results: In our study, 75.8% of the isolates showed phospholipase activity at pH 6.3, and 81 and 97.9% of them showed proteinase activity measured at pH 6.3 and 6.8, respectively. A higher phospholipase activity was detected in strains isolated from dogs with otitis. The proteinase activity was increased at a pH of 6.8 (97.9%) in comparison to a pH of 6.3 (81%)., Conclusions: Our results suggest that the phospholipase activity may play an important role in the invasion of host tissues in chronic canine otitis cases. The proteinase activity results obtained in this study suggest that a reduction in the pH of the treatment may improve its efficacy in the resolution of M. pachydermatis otitis., (Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.)

The strict nutritional requirements of Malassezia species make it difficult to test the antifungal susceptibility. Treatments of the chronic and recurrent infections associated with Malassezia spp. are usually ineffective. The objective of this study was to obtain in vitro susceptibility profile of 76 clinical isolates of Malassezia species against 16 antifungal drugs used for topical or systemic treatment. Isolates were identified by restriction fragment length polymorphism. Minimal inhibitory concentrations (MIC) were obtained by a modified microdilution method based on the Clinical Laboratory Standards Institute reference document M27-A3. The modifications allowed a good growth of all tested species. High in vitro antifungal activity of most tested drugs was observed, especially triazole derivatives, except for fluconazole which presented the highest MICs and widest range of concentrations. Ketoconazole and itraconazole demonstrated a great activity. Higher MICs values were obtained with Malassezia furfur indicating a low susceptibility to most of the antifungal agents tested. Malassezia sympodialis and Malassezia pachydermatis were found to be more-susceptible species than M. furfur, Malassezia globosa, Malassezia slooffiae and Malassezia restricta. Topical substances were also active but provide higher MICs than the compounds for systemic use. The differences observed in the antifungals activity and interspecies variability demonstrated the importance to studying the susceptibility profile of each species to obtain reliable information for defining an effective treatment regimen., (© 2013 Blackwell Verlag GmbH.)

Sertaconazole is a useful antifungal agent against mycoses of the skin and mucosa, such as cutaneous, genital and oral candidiasis and tinea pedis. Its antifungal activity is due to inhibition of the ergosterol biosynthesis and disruption of the cell wall. At higher concentrations, sertaconazole is able to bind to nonsterol lipids of the fungal cell wall, increasing the permeability and the subsequent death of fungal cells. Fungistatic and fungicidal activities on Candida are dose-dependent. The antifungal spectrum of sertaconazole includes deramophytes, Candida, Cryptococcus, Malassezia and also Aspergillus, Scedosporium and Scopulariopsis. Sertaconazole also shows an antimicrobial activity against streptococci, staphylococci and protozoa (Trichomonas). In clinical trials including patients with vulvovaginal candidiasis, a single dose of sertaconazole produced a higher cure rate compared with other topical azoles such as econazole and clotrimazole, in shorter periods. Sertaconazole has shown an anti-inflammatory effect that is very useful for the relief of unpleasant symptoms.

Background: Dermatophytes can be divided into geophilic (soil), zoophilic (animals) and anthropophilic (humans) strains, depending on the source of the keratin that they use for nutritional purposes., Aims: The in vitro susceptibility of clinical isolates of dermatophyte fungi has been studied in the 3 ecological groups with several antifungal agents for the topical management of dermatophytoses in order to determine their relationship with the ecological group., Methods: A standardised dilution micromethod in a liquid medium was used for the determination of the in vitro antifungal activity of 9 topical antifungal drugs: amorolfine (AMR), bifonazole (BFZ), clotrimazole, econazole, ketoconazole, miconazole, oxiconazole, terbinafine (TRB) and tioconazole. The in vitro activity was obtained against 124 clinical isolates of dermatophyte moulds from the anthropophilic, zoophilic and geophilic ecological groups., Results: The in vitro antifungal activity was different depending on the ecological group, although a species-dependent profile was also observed., Conclusions: Azole derivatives showed a similar antifungal profile, being more active against anthropophilic dermatophytes > zoophilic > geophilic. Activity of TRB and AMR was different from that of azole derivatives (zoophilic > anthropophilic > geophilic). A higher in vitro antifungal activity against the 3 ecological groups was observed with TRB and AMR, whilst BFZ was the less active drug., (Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.)

Denture stomatitis is often treated with antifungal agents but recurrences or new episodes are common, and certain episodes can be resistant. New triazoles, such as posaconazole and voriconazole, may represent useful alternatives for management. In vitro activities of amphotericin B, nystatin, miconazole, fluconazole, itraconazole, posaconazole and voriconazole against 150 oral Candida (101 C. albicans, 18 C. tropicalis, 12 C. glabrata, 11 C. guilliermondii, 4 C. parapsilosis, 2 Saccharomyces cerevisiae, 1 C. dubliniensis and 1 C. krusei) from 100 denture wearers were tested by the CLSI M27-A3 method. Resistant isolates were retested by Sensititre YeastOne and Etest. Most antifungal agents were very active. However, 4 C. glabrata (33.3%), 2 C. tropicalis (11.1%), 6 C. albicans (5.6%) and 1 C. krusei were resistant to itraconazole. Posaconazole was active against 143 yeast isolates (95.3%): 6 C. albicans (5.9%) and 1 C. tropicalis (5.6%) were resistant. Geometric mean MICs were 0.036 μg/ml for C. parapsilosis, 0.062 μg/ml for C. albicans, 0.085 μg/ml for C. tropicalis, 0.387 μg/ml for C. guilliermondii and 0.498 μg/ml for C. glabrata. Voriconazole was active against 148 isolates (98.7%) with geometric mean MICs ranging from 0.030 μg/ml for C. parapsilosis, 0.042 μg/ml for C. albicans, 0.048 μg/ml for C. tropicalis, 0.082 μg/ml for C. guilliermondii, to 0.137 μg/ml for C. glabrata. Only 2 C. albicans (2%) were resistant to voriconazole showing cross-resistance to other azoles. Posaconazole and voriconazole have excellent in vitro activities against all Candida isolates and could represent useful alternatives for recalcitrant or recurrent candidiasis.

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., (Copyright © 2012 S. Karger AG, Basel.)

Objectives: There is scarce information on the clinical relevance and antifungal susceptibility of Candida bracarensis, Candida nivariensis, Candida orthopsilosis and Candida metapsilosis. The objective of this study was to assess the prevalence and in vitro antifungal susceptibility of these cryptic species among 173 blood isolates previously identified as Candida glabrata or Candida parapsilosis at the Hospital of Cruces (Barakaldo, Spain). The survey was extended to 518 clinical isolates from the culture collection of the Universidad del País Vasco-Euskal Herriko Unibertsitatea (UPV-EHU; Bilbao, Spain)., Methods: In vitro susceptibilities to 5-fluorocytosine, amphotericin B, anidulafungin, caspofungin, fluconazole, itraconazole, micafungin, posaconazole and voriconazole were tested., Results: All isolates of C. glabrata were identified as C. glabrata sensu stricto. Inside the C. parapsilosis complex, 2.4% of isolates from the Hospital of Cruces and 5.8% from the UPV-EHU were C. metapsilosis or C. orthopsilosis. Of 457 isolates, 435 (95.19%) were C. parapsilosis sensu stricto, 11 (2.41%) C. metapsilosis and 11 (2.41%) C. orthopsilosis. Only seven blood isolates were C. metapsilosis (0.44%) or C. orthopsilosis (1.09%). These cryptic species were also isolated from other relevant clinical specimens. Four C. parapsilosis sensu stricto (5.6%) were susceptible dose-dependent, and one was resistant to both fluconazole and voriconazole (1.4%). Moreover, 19 isolates of C. parapsilosis sensu stricto (26.4%) were intermediately susceptible to itraconazole and higher concentrations of echinocandins were needed to inhibit this species. Most C. orthopsilosis and C. metapsilosis were susceptible to all antifungal agents tested, but one otic isolate of C. metapsilosis was resistant to fluconazole and 5-fluorocytosine., Conclusions: C. metapsilosis and C. orthopsilosis are associated with human disease and show a different antifungal susceptibility profile compared with C. parapsilosis sensu stricto.

The fungistatic and fungicidal activities of sertaconazole against dermatophytes were evaluated by testing 150 clinical isolates of causative agents of tinea pedis, Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton floccosum. The overall geometric means for fungistatic and fungicidal activities of sertaconazole against these isolates were 0.26 and 2.26 μg/ml, respectively, although values were higher for T. mentagrophytes than for the others. This is the first comprehensive demonstration of the fungicidal activity of sertaconazole against dermatophytes.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Nail fungal infections are considered one of the major dermatological problems due to their high rate of therapeutic failure, management and treatment difficulties. Long-term treatments, inadequate therapies, mycological misdiagnosis and follow-up, secondary alterations of the nail, and resistant microorganisms, are some of the causes of these complications. Although the discovery of new antifungal agents has provided some effective molecules, none of the current available drugs are totally effective. It is important to continue researching in this field to provide new antifungal agents and combined therapies., (Copyright 2009 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.)

Tinea capitis is an infection caused by dermatophytes of the genera Microsporum and Trichophyton, and constitutes a major health problem in Argentina. The aim of the present study was to find out the incidence of those etiological agents and the therapeutic response in patients attending a High-Complexity Paediatric Hospital within a two-year period. A total of 98 tinea capitis were diagnosed, 13 of which were Celsus kerion. Microsporum canis was isolated in 61.28%. The range of values for minimum inhibitory concentrations were >32, 0.06-4; <0.015-2; <0.015-0.25; 0.13-8; 0.06-128 microg/mL for fluconazole itraconazole, voriconazole, terbinafine, ketoconazole and griseofulvin, respectively., (Copyright 2009 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.)

Oral candidiasis is the most prevalent complication in HIV-infected and AIDS patients. Topical antifungal treatment is useful for the initial episodes of oral candidiasis, but most patients suffer more than one episode and fluconazole or itraconazole can help in the management, and voriconazole may represent a useful alternative agent for the treatment of recalcitrant oral and oesophageal candidiasis. The aim of this research was to study the in vitro activity of voriconazole and fluconazole against Mexican oral isolates of clinically relevant yeast. The in vitro susceptibility of 187 oral yeast isolates from HIV-infected and healthy Mexicans was determined for fluconazole and voriconazole by the M44-A disc diffusion method. At 24 h, fluconazole was active against 179 of 187 isolates (95.7 %). Moreover, a 100% susceptibility to voriconazole was observed. Voriconazole and fluconazole are highly active in vitro against oral yeast isolates. This study provides baseline data on susceptibilities to both antifungal agents in Mexico.

The in vitro antifungal activity of terbinafine against 521 clinical isolates of seven species of dermatophytes, including four onychomycosis-causative species, as well as five Scopulariopsis brevicaulis isolates was determined by a modified Clinical and Laboratory Standards Institute microdilution method. Results showed a high antifungal activity of terbinafine against all dermatophyte isolates (geometric minimal inhibitory concentration (MIC)=0.026 microg/mL; concentration inhibiting 50% of mycological growth (MIC50)=0.03 microg/mL; and concentration inhibiting 90% of mycological growth (MIC90)=0.06 microg/mL). The geometric mean MICs against onychomycosis-causative dermatophyte species was lower (0.024 microg/mL) than the global MIC. However, the in vitro activity of terbinafine against S. brevicaulis was considerably lower (geometric mean MIC=1.38 microg/mL) in comparison with dermatophytes. The antifungal activity of itraconazole was lower than that of terbinafine against these fungi. These data confirm the high in vitro antifungal activity of terbinafine against dermatophytes, under standardised conditions.

ATB Fungus 2 and SensititreYeastOne are commercial methods for antifungal susceptibility testing of yeasts. The agreement between these two methods was assessed with a total of 133 Candida strains (60 Candida albicans, 18 Candida dubliniensis, 29 Candida glabrata, and 26 Candida krusei). MIC endpoints were established after 24 h of incubation at 36-/+1 degrees C by each method. Intra-laboratory reproducibility of both methods was excellent (=or>99%). Overall agreement between ATB Fungus 2 and Sensititre YeastOne 3 MICs (within 2 dilutions) was 91.2-97.7% for amphotericin B, 5-fluorocytosine and itraconazole, and 82.7% for fluconazole. The categorical agreement when ATB Fungus 2 results were compared to those by SensititreYeastOne 3 was 93.2-98.5% for 5-fluorocytosine and amphotericin B, but lower for the triazoles (72.9-75.9%). This easy to perform method could be an alternative for routine use in the clinical microbiology laboratory for susceptibility testing of common Candida spp.

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., ((c) 2007 S. Karger AG, Basel.)

Voriconazole has been developed to meet the increasing need for new and useful antifungal agents for the treatment of invasive mycoses. This review describes the spectrum of voriconazole antifungal activity based on data from in vitro studies published during the last three years. This survey demonstrates that voriconazole has a broad antifungal spectrum against the most common fungal pathogens being its action fungistatic for Candida and fungicidal for Aspergillus and other filamentous fungi. Overall, more than 95% of all Candida isolates tested are susceptible to voriconazole and less than 3% are resistant. Similar or even better activity rates have been described for Aspergillus, Cryptococcus and most of yeasts and moulds of medical importance. We also discuss the limitations related to the azole cross-resistance observed in some Candida glabrata isolates, the poor activity of voriconazole against Scedosporium prolificans, its activity against fungal biofilms and the great potential usefulness of combination of voriconazole with other antifungal drugs.

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.

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.

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.

Sertaconazole is an imidazole-type antifungal agent that has shown considerable in vitro activity against pathogenic fungi. Various studies carried out in animal models, clinical and toxicologic trials have confirmed the value of sertaconazole in the topical treatment of superficial mycoses in dermatology and gynecology. After several years of clinical experience in the topical treatment of dermatophytosis and Tinea versicolor, the substance has been approved for gynecologic candidiasis in Europe. Sertaconazole has a wide action spectrum that includes yeasts and dermatophyte fungi, and it is also active against bacteria, mainly Gram-positive cocci, making it highly efficient in the treatment of polymicrobial infections. The recent approval of the molecule by the US Food and Drug Administration, and the appearance of a new formulation of sertaconazole for the treatment of onychomycoses on a weekly administrative basis, are all data relevant to the process of marketing the product.

We have studied the in vitro antifungal activity of voriconazole, fluconazole and itraconazole against 252 clinical isolates of dermatophytes and Scopulariopsis brevicaulis by a standardized agar diffusion method (NeoSensitabs). Several important factors such as temperature (28 degrees C vs. 35 degrees C) and incubation time (2-10 days vs. 18-74 h) were adapted to dermatophytes and Scopulariopsis requirements. Voriconazole showed an excellent activity against most species of dermatophytes, higher than itraconazole and fluconazole. However, S. brevicaulis isolates were highly resistant to all azoles used in this study. Voriconazole might be an interesting antifungal alternative to refractory superficial mycoses.

Objectives: The in vitro antifungal activity of posaconazole was compared with that of fluconazole and amphotericin B., Materials and Methods: A microdilution method (M27-A2) was used with 331 clinical yeast isolates., Results: The geometric mean MICs of posaconazole, fluconazole and amphotericin B were 0.16, 0.91 and 0.15 mg/L, respectively. Posaconazole was markedly more active than fluconazole and was active against 9/11 fluconazole-resistant Candida albicans, and five Candida glabrata had an MIC of posaconazole of 16 mg/L., Conclusions: These data indicate that posaconazole is a potentially effective antifungal agent for the treatment of mycoses caused by yeasts.

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., (2004 S. Karger AG, Basel.)

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.

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.

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.

The aim of this study was to determine the prevalence of primary resistance to 5-fluorocytosine (5FC) among clinical isolates of yeasts in Spain where this drug is not currently available for therapy. We have tested the in vitro activity of 5FC against 1,021 recent yeast clinical isolates, including 522 Candida albicans, 140 Candida parapsilosis, 68 Candida glabrata, 41 Candida dubliniensis, 50 Candida guilliermondii, 34 Candida tropicalis, 28 Candida krusei, 20 Candida famata, 11 Cryptococcus neoformans, 5 Cryptococcus albidus, 43 Rhodotorula spp., 24 Trichosporon spp., 5 Saccharomyces cerevisiae, 9 Pichia spp., and 21 isolates from other 11 yeast species. The MICs were determined by the ATB Fungus agar microdilution test (bioMerieux, France) and the following interpretive breakpoints were used: susceptible, > 4 microg/ml; intermediate, 8 to 16 microg/ml; resistant, > 32 microg/ml. 5FC was very active against Candida spp. and other medically important yeasts as 852 (83.4%) of the studied isolates were susceptible (MIC < 4 microg/ml). The species most susceptible to 5FC were C. dubliniensis (100%of isolates; MIC90, 0.25 microg/ml), C. famata (100% of isolates; MIC90, 0.25 microg/ml), C. guilliermondii (98%of isolates; MIC90, 0.25 microg/ml), C. glabrata (95.5% of isolates; MIC90, 0.25 microg/ml), and C. neoformans (90.9% of isolates; MIC90, 2 microg/ml). Primary resistance to 5FC was very uncommon, and a MIC > 32 microg/ml, indicator of in vitro resistance, was observed in 106 isolates (10.4%): 77 C. albicans (16.5% of isolates; MIC90, > 128 microg/ml), 9 C. parapsilosis (6.4% of isolates; MIC90, 8 microg/ml), 4 C. albidus (80% of isolates, MIC50, > 128 microg/ml), 3 C. glabrata (4.4% of isolates; MIC90, 0.25 microg/ml), 3 C. tropicalis (8.8% of isolates; MIC90, 4 microg/ml), 2 C. krusei (7.1% of isolates; MIC90, 8 microg/ml), 2 Rhodotorula spp. (4.6% of isolates, MIC90, 1 microg/ml), 8 Trichosporon spp. (33.3% of isolates; MIC90, 64 microg/ml), and 1 C. lipolytica (50% of isolates). Interestingly, most C. albicans (67 out of 77 isolates) resistant to 5FC were serotype B isolates.

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.

Three hundred and nine strains belonging to 11 species of dermatophyte moulds were tested against sertaconazole following mainly the National Committee for Clinical Laboratory Standards (M38-P) for filamentous fungi. However, several important factors such as the temperature (28 degrees C vs 35 degrees C) and time of incubation (4-10 d vs 21-74 h), have been modified. Sertaconazole was active against all the clinically important dermatophyte moulds involved in human infections tested. Overall geometric mean MIC of sertaconazole was 0.21 microg/ml with a MIC range of 0.01-8 microg/ml. MIC50 and MIC90 were respectively of 0.25 and 1 microg/ml. Sertaconazole was very active against Epidermophyton floccosum, Trichophyton rubrum, Trichophyton tonsurans and Microsporum canis (geometric means 0.08, 0.13, 0.13 and 0.19 microg/ml respectively). Microsporum audouinii had the lowest susceptibility in the study (geometric mean 0.59 microg/ml). Considering MIC50 and MIC90 these differences were significantly in favor of the activity of sertaconazole against E. floccosum (0.06 and 0.5 microg/ml respectively).

We have studied the in vitro antifungal activity of sertaconazole against 114 dermatophytes with low susceptibility to fluconazole following the National Committee for Clinical Laboratory Standards for filamentous fungi (M38-P). However, several important factors such as the temperature (28 vs. 35 degrees C) and time of incubation (4-10 days vs. 21-74 h), have been found to affect dermatophytes. Isolates were recently recovered from human samples. Sertaconazole was active against 114 isolates of 12 fungal dermatophyte species, showing an overall geometric mean of 0.41 microg/ml with a minimum inhibitory concentration (MIC) range of 0.01-2 microg/ml against these isolates with reduced fluconazole susceptibility. Differences between both antifungals were significant (p < 0.05). MIC(50) and MIC(90) of sertaconazole were of 0.5 and 1 microg/ml, respectively, while the MIC of fluconazole was >/=16 microg/ml. None of the isolates was resistant to sertaconazole during the study while for four isolates the MIC of fluconazole was >/=64 microg/ml. No evidence of cross-resistance between both antifungals was found., (Copyright 2003 S. Karger AG, Basel)

This study further evaluated the in vitro activity of anidulafungin (VER002, Versicor Inc.) (LY303366) against 460 clinical yeast isolates. MICs of anidulafungin, fluconazole and itraconazole were determined by following the NCCLS M27-A guidelines. Minimum fungicidal concentrations (MFCs) of anidulafungin were determined for 230 isolates of Candida spp. The activity of anidulafungin in vitro was significantly superior (P < 0.05) to those of itraconazole and fluconazole against Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei, but anidulafungin was less active for Candida famata and Candida parapsilosis. The differences were not significant for the other species evaluated.

We compared the in vitro antifungal activity of amphotericin B lipid complex (ABLC) with that of itraconazole (ITZ) against 535 yeast strains and 173 opportunistic filamentous fungi by using a microdilution method (National Committee for Clinical Laboratory Standards M27-A and M38-P). The overall geometric mean MIC was 0.13 microg/ml and 0.177 microg/ml for ITZ and ABLC, respectively, and the MIC(50) was 0.125 microg/ml for both agents against yeast isolates. ITZ had a similar or slightly superior efficacy compared to ABLC when tested against Candida albicans, Candida parapsilosis, Cryptococcus neoformans, Candida krusei, Candida glabrata and Candida tropicalis. Effectiveness against C. glabrata was lower for ITZ (MIC(90) 2 microg/ml, and for ABLC, 0.5 microg/ml). For Aspergillus fumigatus, activity of ITZ was superior in comparison with ABLC (MIC(90) 1 and 16 microg/ml, respectively); MIC(90) for Aspergillus niger was 4 and 2 microg/ml for ABLC and ITZ, respectively. Scedosporium spp. showed a low susceptibility to both ABLC and ITZ. In conclusion, ABLC and ITZ are useful alternatives for the treatment of severe fungal infections. The selection of an antifungal agent should be made considering the toxicological and pharmacological properties and cost/benefit relationship and be supported by the susceptibility of the isolate., (Copyright 2002 S. Karger AG, Basel)