A collection of Aspergillus fumigatus mutants highly resistant to itraconazole (RIT) at 100 g ml ؊1 were selected in vitro (following UV irradiation as a preliminary step) to investigate mechanisms of drug resistance in this clinically important pathogen. Eight of the RIT mutants were found to have a mutation at Gly54 (G54E, -K, or -R) in the azole target gene CYP51A. Primers designed for highly conserved regions of multidrug resistance (MDR) pumps were used in reverse transcriptase PCR amplification reactions to identify novel genes encoding potential MDR efflux pumps in A. fumigatus. Two genes, AfuMDR3 and AfuMDR4, showed prominent changes in expression levels in many RIT mutants and were characterized in more detail. Analysis of the deduced amino acid sequence encoded by AfuMDR3 revealed high similarity to major facilitator superfamily transporters, while AfuMDR4 was a typical member of the ATP-binding cassette superfamily. Real-time quantitative PCR with molecular beacon probes was used to assess expression levels of AfuMDR3 and AfuMDR4. Most RIT mutants showed either constitutive high-level expression of both genes or induction of expression upon exposure to itraconazole. Our results suggest that overexpression of one or both of these newly identified drug efflux pump genes of A. fumigatus and/or selection of drug target site mutations are linked to high-level itraconazole resistance and are mechanistic considerations for the emergence of clinical resistance to itraconazole.

Antifungal efficacies of the echinocandin drugs caspofungin, micafungin, and anidulafungin were reduced significantly in the presence of 50% human serum, which yielded nearly equivalent MICs or minimum effective concentrations against diverse Candida spp. and Aspergillus spp. Consistent with a direct drug interaction, serum decreased the sensitivity of glucan synthase to echinocandin drugs.The echinocandin drugs caspofungin, micafungin, and anidulafungin inhibit the fungal ␤-1,3-glucan synthase enzyme, which blocks the formation of glucan polymers, thereby disrupting fungal cell wall integrity (2). Animal and human studies indicate that echinocandin drugs are extensively bound to serum proteins (1,4,5,15), and serum was shown to reduce the antifungal properties of micafungin with some Candida spp. (3), yet little is known about the influence of serum on antifungal efficacy with the different echinocandin drugs.Echinocandin susceptibility in the presence or absence of 50% human serum (Sigma) was evaluated with a diverse collection of clinical isolates, laboratory strains, and reference strains of Candida spp. and Aspergillus spp., according to the guidelines in CLSI documents M27-A2 (9) and M38-A (8), respectively. Abnormal colony morphology was used to establish a minimum effective concentration (MEC) for Aspergillus spp. after 48 h of incubation at 35°C (6). Glucan synthase (GS) isolation and 50% inhibitory concentration (IC 50 ) inhibition kinetics were performed as described previously (11). A murine candidiasis model utilizing female BALB/c mice (age, 10 to 12 weeks; weight, 20 to 25 g) was used to assess the relative in vivo efficacies of echinocandin drugs (13).Serum increased caspofungin MICs an average of 2-fold, with a range of 1-to 16-fold, while it had a more pronounced effect on the other drugs, increasing the MIC an average of 16-fold with a range of 8-to 256-fold for anidulafungin and an average of 64-fold with a range of 32-to 128-fold for micafungin (Table 1). The effects of serum on MICs were assessed for other non-Candida albicans spp. The largest MIC shift for caspofungin (eightfold) was with Candida krusei, while Candida tropicalis strains showed the most significant shifts (128-fold) for both micafungin and anidulafungin. These drugs consistently showed pronounced shifts, which reflected their greater antifungal potencies in the absence of serum. These differences disappeared in the presence of 50% serum, where all three drugs showed comparable MICs.Similar serum-induced effects were observed with a collection of Aspergillus spp., where microscopically observed MECs for the three drugs shifted higher in the presence of serum. Micafungin and anidulafungin again showed the most pronounced antifungal shifts, 32-to 133-fold and 16-to 32-fold, respectively, reflecting their more active behavior in the absence of serum ( Table 2). The three drugs displayed nearly equivalent MECs in the presence of 50% serum. The reduced antifungal properties of echinocandin drugs in the presence of serum suggested...

Amphotericin B (AMB) remains the principal therapeutic choice for deep mycoses. However, its application is limited by toxicity and a route of administration requiring slow intravenous injection. An oral formulation of this drug is desirable to treat acute infections and provide prophylactic therapy for high-risk patients. Cochleates are a novel lipid-based delivery system that have the potential for oral administration of hydrophobic drugs. They are stable phospholipid-cation crystalline structures consisting of a spiral lipid bilayer sheet with no internal aqueous space. Cochleates containing AMB (CAMB) inhibit the growth of Candida albicans, and the in vivo therapeutic efficacy of CAMB administered orally was evaluated in a mouse model of systemic candidiasis. The results indicate that 100% of the mice treated at all CAMB doses, including a low dosage of 0.5 mg/kg of body weight/day, survived the experimental period (16 days). In contrast, 100% mortality was observed with untreated mice by day 12. The fungal tissue burden in kidneys and lungs was assessed in parallel, and a dose-dependent reduction in C. albicans from the kidneys was observed, with a maximum 3.5-log reduction in total cell counts at 2.5 mg/kg/day. However, complete clearance of the organism from the lungs, resulting in more than a 4-log reduction, was observed at the same dose. These results were comparable to a deoxycholate AMB formulation administered intraperitoneally at 2 mg/kg/day (P < 0.05). Overall, these data demonstrate that cochleates are an effective oral delivery system for AMB in a model of systemic candidiasis.The opportunistic fungal pathogen Candida albicans causes life-threatening infections among cancer patients, organ or bone marrow transplant recipients, and patients with congenital and acquired immunodeficiencies (4,14,17,24,28,29,32,43). Candida is the fourth leading cause of nosocomial bloodstream infections, accounting for 8% of all infections, and remains an important cause of morbidity and mortality in immunocompromised patients (7,28,31,32,43).Parenteral administration of amphotericin B (AMB), a polyene antibiotic with strong antifungal activity, remains the therapy of choice for systemic mycoses. It is highly hydrophobic and is commonly administrated as desoxycholate amphotericin (DAMB), a detergent micelle complex. AMB binds preferentially to ergosterol in fungal plasma membranes, although it also interacts with animal cell sterols such as cholesterol, which accounts for known toxicity (10, 35). DAMB therapy is associated with nephrotoxicity, central nervous system and liver damage, and side effects such as nausea and fever (23,34,35). AMB, with its inherent low solubility in water and many organic solvents, shows relatively poor bioavailability (11,12). In order to increase the therapeutic index of AMB and reduce its associated toxicity, new lipid-based formulations have been developed (1,3,33). These drug delivery systems, such as liposomal formulations, lipid complexes, lipid emulsions, and colloidal dispersion...

Cochleates containing amphotericin B (CAMB) were administered orally at doses ranging from 0 to 40 mg/kg of body weight/day for 14 days in a murine model of systemic aspergillosis. The administration of oral doses of CAMB (20 and 40 mg/kg/day) resulted in a survival rate of 70% and a reduction in colony counts of more than 2 logs in lungs, livers, and kidneys. Orally administered CAMB shows promise for the treatment of aspergillosis.Aspergillus fumigatus causes a variety of diseases, including allergic bronchopulmonary aspergillosis in asthma patients and invasive pulmonary aspergillosis in immunocompromised patients (1, 7). Invasive pulmonary aspergillosis can be treated with broad-spectrum triazoles (2, 14) and echinocandins (14), yet amphotericin B (AmB) therapy remains the preferred treatment for severe Aspergillus infections (12). Unlike other classes of antifungals, AmB is highly toxic and shows poor oral bioavailability. In order to increase the therapeutic index of AmB, new lipid-based formulations have been developed (4,5,8). Despite improvement in the therapeutic index for liposomal AmB formulations, the overall prognosis for patients with invasive disease remains poor. Recently, cochleate delivery vehicles for amphotericin B have been introduced as a new platform for overcoming the poor oral bioavailability of AmB (9,16,17). It has been shown that orally administered cochleates containing amphotericin B (CAMB) were as effective as intraperitoneally (i.p.) administered deoxycholate AmB (DAMB) in protecting against mortality and reducing the fungal burden of tissues in a murine model of candidiasis (9). In this paper, we describe the efficacy in vivo of CAMB delivered orally in a mouse model of systemic aspergillosis.CAMB were prepared by use of an aqueous/aqueous hydrogel binary system (9) and produced an MIC of Ͻ1 g/ml for A. fumigatus challenge strain MSKCC R21 when tested in RPMI 1640 medium according to NCCLS protocol M38-P. CAMB were evaluated in a systemic aspergillosis model adapted from that described by Verweij and colleagues (13). Female BALB/c mice (20 to 22 g) were rendered susceptible to A. fumigatus infection by treatment with cyclophosphamide at 150 to 200 mg/kg of body weight via intravenous injection through the lateral tail vein 3 days prior to infection with 0.1 ml of saline containing 10 6 spores of A. fumigatus (MSKCC R21). Treatment was initiated immediately after infection by oral administration of CAMB for 14 days at 0 to 20 mg/kg/day for the group treated with 150 mg of cyclophosphamide/kg and at 0 to 40 mg/kg/day for the group treated with 200 mg of cyclophosphamide/kg. DAMB administered i.p. at 4 mg/kg/day was used as a positive control. Survival rates and tissue burden (CFU per gram) in kidneys, livers, and lungs were determined for each treatment.The efficacy in vivo of orally administered CAMB was evaluated in two separate trials of the disseminated aspergillosis model in which mice (10 per group) were treated daily for 14 days. In trial 1 (Fig. 1A), immunosuppression was ...

Cryptococcus neoformans is an AIDS-associated human fungal pathogen and the most common cause of fungal meningitis, with a mortality rate over 40% in AIDS patients. Significant advances have been achieved in understanding its disease mechanisms. Yet the underlying mechanism of a high frequency of cryptococcal meningitis remains unclear. The existence of high inositol concentrations in brain and our earlier discovery of a large inositol transporter (ITR) gene family in C. neoformans led us to investigate the potential role of inositol in Cryptococcus-host interactions. In this study, we focus on functional analyses of two major ITR genes to understand their role in virulence of C. neoformans. Our results show that ITR1A and ITR3C are the only two ITR genes among 10 candidates that can complement the growth defect of a Saccharomyces cerevisiae strain lacking inositol transporters. Both S. cerevisiae strains heterologously expressing ITR1A or ITR3C showed high inositol uptake activity, an indication that they are major inositol transporters. Significantly, itr1a itr3c double mutants showed significant virulence attenuation in murine infection models. Mutating both ITR1A and ITR3C in an ino1 mutant background activates the expression of several remaining ITR candidates and does not show more severe virulence attenuation, suggesting that both inositol uptake and biosynthetic pathways are important for inositol acquisition. Overall, our study provides evidence that host inositol and fungal inositol transporters are important for Cryptococcus pathogenicity.