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.

Heterogeneous mechanisms of azole resistance in Candida albicans clinical isolates from an HIV-infected patient on continuous fluconazole therapy for oropharyngeal candidosis.

Molecular mechanisms of azole resistance in Candida albicans include alterations in the target enzyme and increased efflux of drug, but the impact of specific treatment regimens on resistance has not been established. A patient with advanced AIDS was enrolled in a longitudinal study to receive continuous oral fluconazole (FLU) 200 mg/day for the treatment of oropharyngeal candidosis (OPC). Oral cultures were obtained at time of enrollment, during episodes of OPC and quarterly for surveillance. The patient had five symptomatic relapses on continuous FLU during 43 months. All OPC episodes were successfully treated with increasing doses of FLU although increased FLU MICs were detected for C. albicans isolates with progression of time. DNA-typing techniques demonstrated that resistance developed in a persistent strain of C. albicans. Both FLU-resistant and isogenic isolates with reduced susceptibility were detected in the same clinical samples through multiple episodes. Analysis of molecular mechanisms of resistance revealed overexpression of MDR and CDR genes encoding efflux pumps (but not ERG11) in isolates with decreased FLU susceptibility. In addition, the presence of the G464S amino acid substitution in their lanosterol demethylase, affecting its affinity for FLU, was also detected. However, other isogenic, but FLU-susceptible isolates recovered from the same samples did not harbour the mutation, indicating microevolution of yeast populations within the oral cavity. In this patient, the continuous antifungal pressure exerted by FLU resulted in development of resistance of multifactorial nature. Despite their clonal origin, different subpopulations of C. albicans demonstrated distinct resistance mechanisms, including concomitant presence and absence of functional point mutations in ERG11 genes.

[Activity of itraconazole against clinical isolates of Aspergillus spp. and Fusarium spp. determined by the M38-P NCCLS method]. / Actividad del itraconazol frente a aislamientos clínicos de Aspergillus spp. y Fusarium spp. determinada por el método M38-P del NCCLS.

The antifungal activity of itraconazole was studied in 101 clinical isolates of Aspergillus fumigatus, A. flavus, A. niger, A. terreus, A. nidulans, A. candidus, A. glaucus, A. clavatus, Fusarium solani, F. oxysporum and F. semitectum. The minimum inhibitory concentrations (MIC) were determined according to the protocol of the M38-P National Committee for Laboratory Standards (NCCLS) document using a microdilution method in 1640 RPMI liquid medium (visual reading at 48 and 72 h incubation). In general, the MIC did not vary with time of incubation, except in a Z. fumigatus strain in which the MIC went from 2 to 16 mg/l. The geometric mean of the MIC and MIC(90) of itraconazole for Aspergillus spp. was 0.44 mg/l and 0.5 mg/l, respectively; and for Fusarium spp. it was 14.1 mg/l and 16 mg/l, respectively. With 0.5 mg/l 75% of the Aspergillus spp. strains were inhibited, and 100% of these strains were inhibited with 2 mg/l. A. niger and A. fumigatus were the most resistant species (MIC(90) 2 mg/l). The MIC of all the Fusarium strains essayed was between 4 and 16 mg/l.

Actividad del itraconazol frente a aislamientos clínicos de Aspergillus spp. y Fusarium spp. determinada por el método M38-P del NCCLS / Activity of itraconazole against clinical isolates of Aspergillus spp. and Fusarium spp. determined by the M38-P NCCLS method

Se ha estudiado la actividad antifúngica del itraconazol en 101 aislamientos clínicos de Aspergillus fumigatus, A. flavus, A. niger, A. terreus, A. nidulans, A. candidus, A. glaucus, A. clavatus, Fusarium solani, F. oxysporum y F. semitectum. Las concentraciones mínimas inhibitorias (CMI) se determinaron siguiendo el protocolo del documento M38-P del NCCLS por medio de un método de microdilución en medio líquido RPMI 1640 (lectura visual a las 48 y 72 horas de incubación). En general, la CMI no varió con el tiempo de incubación, excepto en una cepa de A. fumigatus en la cual la CMI pasó de 2 a 16 mg/l. La media geométrica de las CMI y la CMI90 de itraconazol para Aspergillus spp. fueron 0,44 mg/l y 0,5 mg/l, respectivamente, y para Fusarium spp. 14,1 mg/l y 16 mg/l, respectivamente. Con 0,5 mg/l se inhibió el 75 por ciento de las cepas de Aspergillus spp., y con 2 mg/l el 100 por ciento de ellas. A. niger y A. fumigatus fueron las especies más resistentes (CMI90 2 mg/l). Las CMI de todas las cepas de Fusarium ensayadas estuvieron comprendidas entre 4 y 16 mg/l (AU)

In vitro susceptibility of Candida dubliniensis to current and new antifungal agents.

BACKGROUND: Candida dubliniensis is a recently described Candida species closely related to Candida albicans, which has been associated with oral candidiasis in HIV-infected patients. Fluconazole-resistant strains of C. dubliniensis are easily obtained in vitro and this fact could be a complication if this resistance develops during treatment with this drug. METHODS: In the present study, the in vitro antifungal susceptibilities of 36 C. dubliniensis clinical isolates and culture strains to current and new antifungal agents, such as amphotericin B (AMB), amphotericin B lipid complex (ABLC), amphotericin B colloidal dispersion (ABCD), 5-fluorocytosine (5FC), fluconazole (FLC), itraconazole (ITC), ketoconazole (KTC), liposomal amphoteri- cin B (LAMB), liposomal nystatin (LNYT), LY303366 (LY), SCH56592 (SCH), and voriconazole (VRC), were determined according to the National Committee for Clinical Laboratory Standards M27-A broth microdilution method for yeasts. RESULTS: Most isolates of C. dubliniensis were susceptible to both new and current antifungal drugs, with 75.9% isolates susceptible to KTC, 86.2% to FLC and to ITC, and approximately 100% to the other antifungal agents tested. The cross-resistance phenotypes are detailed. Four isolates were resistant (MIC > or =64 microg/ml) to FLC. These 4 isolates were also resistant to KTC, and 3 of them were also resistant to ITC (MIC > or =1 microg/ml for both agents). However, these isolates were highly susceptible to 5FC and all polyene formulations (AMB, ABLC, ABCD, LAMB, and LNYT), triazole (SCH and VRC) and echinocandin (LY) antifungal agents. CONCLUSION: The new liposomal and lipidic formulations of AMB, LNYT, and the new triazoles and echinocandins may provide new alternatives to FLC for the treatment of infections by C. dubliniensis.

Comparative in vitro antifungal activity of amphotericin B lipid complex, amphotericin B and fluconazole.

Amphotericin B (AMB) is considered the gold standard in the treatment of serious systemic mycoses in spite of its nephrotoxicity and adverse effects. Association with lipids enables larger doses of AMB to be given with a longer t((1/2)) and C(max), without the toxic effects at lower concentrations. Liposome-encapsulated AMB shows a lower affinity for mammalian cells and improves V(d), thus decreasing toxicity. Amphotericin B lipid complex (ABLC) is an AMB formulation associated with a biodegradable phospholipid matrix (5% molar) from which the drug is released by cell phospholipases. ABLC is recommended for serious mycoses refractory to conventional antifungal therapy or when AMB is contraindicated. We compared the in vitro antifungal activity of ABLC, AMB and fluconazole (FLZ) against 328 strains of clinically significant opportunistic fungi using a microdilution method (NCCLS, M-27A). 64.9% of the yeasts were inhibited by MIC of ABLC

[In vitro activity of a liposomal nystatin formulation (Nyotran) against Cryptococcus neoformans]. / Actividad in vitro de una formulación liposómica de nistatina (Nyotran) frente a Cryptococcus neoformans.

The in vitro antifungal activity of a new liposomal nystatin formulation (NISTL, Nyotran, Aronex Ltd., EE.UU.) was evaluated by a microdilution method with RPMI based on the M27A document of the National Committee for Clinical Laboratory Standards (NCCLS) against 22 isolates of Cryptococcus neoformans. This antifungal activity was compared with those of other seven antifungal agents, such as nystatin (NIST), amphotericin B deoxycholate, liposomal amphotericin B, amphotericin B lipid complex, amphotericin B colloidal dispersion, fluconazole, and itraconazole. NISTL was more active in vitrothan NIST, showing MIC values 2-3 fold smaller in 90% of the isolates. The results obtained suggest that this new formulation would be very helpful for the treatment of cryptococcosis.

[Determination of the in vitro antifungal susceptibility of clinically important yeasts using the Sensititre system]. / Determinación mediante el sistema Sensititre de la sensibilidad in vitro a los antifúngicos de levaduras de interés médico.

Using Sensititre (AccuMed, USA) we studied the in vitro antifungal activity of amphotericin B, fluconazole, itraconazole, ketoconazole and 5-fluorocytosine against 250 clinical yeast isolates taken from different hospitals, including Candida (151 C. albicans, 15 C. krusei, 14 C. parapsilosis, 11 C. tropicalis, 10 C. glabrata, 4 C. guilliermondii, 3 C. rugosa, 2 C. viswanathii, 2 C. famata and 2 C. kefyr), Cryptococcus (32 C. neoformans and 1 C. laurentii), Trichosporon (2 isolates) and Rhodotorula rubra (1 isolate). All the strains were susceptible to amphotericin B and showed an MIC <1 mg/l. The susceptibility of C. albicans (MIC(90) <256 mg/l), C. krusei (MIC(90) <64 mg/l), C. glabrata (MIC(90) <64 mg/l) and C. neoformans (MIC(90) 32 mg/l) to fluconazole was lower (14% isolates being resistant and 16.8% susceptible depending on the dose). The largest number of strains resistant to itraconazole was observed in C. albicans and C. glabrata (17.2% resistant and 24% susceptible and susceptible depending on the dose, respectively). Ketoconazole and 5-fluorocytosine were not effective in vitro against 12.8% and 2%, respectively, of all the isolates studied. Nine C. krusei and seven C. neoformans (12.9%) showed dose-dependent susceptibility to 5-fluorocytosine.

In-vitro antifungal activity of liposomal nystatin in comparison with nystatin, amphotericin B cholesteryl sulphate, liposomal amphotericin B, amphotericin B lipid complex, amphotericin B desoxycholate, fluconazole and itraconazole.

The in-vitro susceptibilities of 120 clinical isolates of yeasts to liposomal nystatin were compared with those to amphotericin B lipid complex (ABLC), liposomal amphotericin B (LAB), amphotericin B cholesteryl sulphate (ABCD), amphotericin B desoxycholate, nystatin, fluconazole and itraconazole. Yeast isolates examined included strains of Candida albicans, Candida parapsilosis, Candida glabrata, Candida krusei, Candida guilliermondii, Candida tropicalis, Candida kefyr, Candida viswanathii, Candida famata, Candida rugosa, Rhodotorula rubra, Trichosporon spp., Cryptococcus laurentii and Cryptococcus neoformans. The mean MICs for all strains examined were: liposomal nystatin 0.96 mg/L; nystatin 0.54 mg/L; ABLC 0.65 mg/L; LAB 1.07 mg/L; ABCD 0.75 mg/L; amphotericin B 0.43 mg/L; fluconazole 5.53 mg/L; and itraconazole 0.33 mg/L. No significant differences were seen between the activity of liposomal nystatin and the polyene drugs or itraconazole, but liposomal nystatin was more active than fluconazole. MICs were lower than the reported blood concentrations following therapeutic doses of this drug, indicating the potential for a therapeutic use of liposomal nystatin in humans. These results indicate good activity in vitro against medically important yeasts, which compares favourably with the activities of other currently available antifungal drugs. Liposomal nystatin may have a role in the treatment of disseminated and systemic mycoses.

[Prevalence of oral lesions by Candida sp.: Their varieties and serotypes in a population of patients with AIDS under a highly active antiretroviral therapy.]. / Prevalencia de lesiones orales por Candida en una población con sida sometida a terapia antirretroviral altamente activa.

The aim of this study has been to determine the prevalence of oral candidiasis and oral Candida carriers in an AIDS population under highly active antiretroviral therapy. Eighty-six AIDS patients treated with an antiretroviral combination (indinavir o ritonavir o saquinavir + zidovudine [AZT] + lamivudine [3TC]). Patients were grouped attending the predisposing factors for HIV infection in: intravenous drug users (IDU), heterosexuals, homosexuals, patients using hematological products or having unknown factors. Oral cavity was examined and an oral specimen was inoculated in a chromogenic culture medium (Albicans ID, bioMérieux, France). The prevalence of oral Candida lesions was 30.2% and Candida was isolated from 54.7% of patients. The predominant species was C. albicans serotype A in all the groups with the exception of homosexual patients, were C. albicans serotype B was the predominant. The IDU group showed the higher prevalence of Candida lesions and oral yeasts colonization, followed by the group of heterosexuals and homosexuals. An association was found between the presence of lesions and/or Candida spp. and the clinical stage or the viral concentration. The species Candida dubliniensis was isolated in the oral samples of two patients with candidosis and in two individuals without oral candidosis. The finding of this species in Spanish patients can be added to the data obtained in epidemiological studies in other countries.