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.     

Activities of triazole-echinocandin combinations against Candida species in biofilms and as planktonic cells

Biofilm formation complicates the treatment of various infections caused by Candida species. We investigated the effects of simultaneous or sequential combinations of two triazoles, voriconazole (VRC) and posaconazole (PSC), with two echinocandins, anidulafungin (AND) and caspofungin (CAS), against Candida albicans and Candida parapsilosis biofilms in comparison to their planktonic counterparts. Antifungal activity was assessed by the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide (XTT) metabolic assay. Antifungal-agent interactions were analyzed by the Bliss independence model in the simultaneous-treatment studies and by analysis of variance (ANOVA) in the sequential-treatment studies. Against C. albicans planktonic cells, the simultaneous combination of PSC (32 to 128 mg/liter) and CAS (0.008 to 0.25 mg/liter) was synergistic; the combinations of PSC (128 to 1,024 mg/liter) with AND (0.03 to 0.5 mg/liter) and VRC (32 to 512 mg/liter) with AND (0.008 to 0.03 mg/liter) were antagonistic. Against C. parapsilosis planktonic cells, the interaction between VRC (32 to 1,024 mg/liter) and CAS (1 to 16 mg/liter) was antagonistic. All simultaneous antifungal combinations demonstrated indifferent interactions against biofilms of both Candida species. Damage to biofilms of both species increased (P<0.01) in the presence of subinhibitory concentrations of echinocandins (0.008 to 0.064 mg/liter), followed by the addition of PSC (512 mg/liter for C. albicans and 64 to 512 mg/liter for C. parapsilosis) or VRC (256 to 512 mg/liter for C. albicans and 512 mg/liter for C. parapsilosis). Triazole-echinocandin combinations do not appear to produce antagonistic effects against Candida sp. biofilms, while various significant interactions occur with their planktonic counterparts.     

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 相似文献   

Fungal phospholipase activity and susceptibility to lipid preparations of amphotericin B

It has been postulated that phospholipases of fungal origin can affect in vitro susceptibility testing of amphotericin B lipid complex (ABLC). We used specific phospholipase-deficient mutants of Candida albicans and Cryptococcus neoformans in susceptibility testing and demonstrated that extracellular fungal phospholipase activity does not influence the in vitro susceptibilities of these two fungi to ABLC.     

Pharmacokinetic-pharmacodynamic comparison of amphotericin B (AMB) and two lipid-associated AMB preparations, liposomal AMB and AMB lipid complex, in murine candidiasis models

It is generally accepted that the lipid formulations of amphotericin B (AMB) are not as potent as conventional AMB on a milligram-per-kilogram basis. We used a neutropenic murine disseminated candidiasis model to compare the in vivo potencies of AMB, liposomal AMB (L-AMB), and AMB lipid complex (ABLC) pharmacodynamically. The pharmacokinetics of the antifungals were examined in serum and in three organs commonly seeded in disseminated candidiasis (kidneys, liver, and lung). Both single-dose time-kill studies and multiple-dosing-regimen studies were used with each of the compounds. Determinations of the numbers of CFU in the kidneys were performed following the administration of three escalating single doses of the polyenes at various times over 48 h. The areas under the time-kill curves (AUTKs) for each dose level of the drugs were compared by analysis of variance (ANOVA). In the multiple-dosing-regimen studies with five Candida isolates, AMB, L-AMB, and ABLC were administered daily for 72 h. The organism burdens in the mouse kidneys were similarly used as the treatment end point. Additional multiple regimen-dosing-studies were performed with a single Candida albicans isolate, and the microbiologic outcomes in four internal organs (kidneys, liver, spleen, and lung) were examined at the end of therapy (48 h). The relationship between the dose and the drug exposure expressed by the pharmacokinetics of the dosing regimens in serum and organ tissue were analyzed by using a maximum-effect model. ANOVA was used to compare the drug exposures necessary to achieve the 25% effective dose (ED25), ED50, ED75, and 1 log10 killing. Comparison of AUTKs suggested that AMB was 4.3- to 5.9-fold more potent than either ABLC or L-AMB. The time-kill curves for both lipid formulations were very similar. In the multiple-dosing-regimen studies, AMB was 5.0- to 8.0-fold more potent than each of the lipid formulations against five Candida isolates in the kidneys. Similar differences in potency (5.1- to 7.2-fold) were observed in the other end organs. The difference in pharmacokinetics in serum accounted for much of the difference in potency between AMB and ABLC (ratio of serum ABLC area under the curve of effective doses to serum AMB area under the curve of effective doses, 1.2). The differences in the kinetics in the various end organs between AMB and L-AMB were better at explaining the disparate potencies at these infection sites (ratio of organ L-AMB area under the curve of effective doses to organ AMB area under the curve of effective doses, 1.1).     

Influence of phospholipid/amphotericin B ratio and phospholipid type on in vitro renal cell toxicities and fungicidal activities of lipid-associated amphotericin B formulations.

We studied the influence of the lipid/amphotericin B (AMB) ratio and the phospholipid type on the in vitro renal cell toxicity and antifungal efficacy of lipid-associated AMB (L-AMB). L-AMB was prepared at one of two different lipid/AMB ratios (1 and 40) by incubating AMB with empty small unilamellar vesicles, made from one of three different phospholipids: dipalmitoyl-, dimirystoyl-, and distearoylphosphatidylcholine (DPPC, DMPC, and DSPC, respectively). Renal cell toxicity, investigated through an assessment of the Na-dependent uptake of phosphate by proximal tubular cells, and fungicidal effect against Candida albicans were studied after 1 h of treatment at 37 degrees C. The amount of unbound AMB present in each L-AMB formulation was studied by use of circular dichroism. At a lipid/AMB ratio of 40, the three lipidic formulations were not toxic for renal cells but were less effective against C. albicans than AMB; however, DSPC-AMB, which contained 50% unbound AMB, was more effective against C. albicans than DPCC-AMB or DMPC-AMB, containing 0 and 13% unbound AMB, respectively. At a lipid/AMB ratio of 1, the antifungal effects of L-AMB and AMB were similar, whatever the phospholipid used, but only DMPC-AMB remained highly protective against AMB renal cell toxicity, despite the presence of the same amount of unbound AMB (50%) in DMPC-AMB and DPPC-AMB. We conclude that the in vitro activities and renal cell toxicities of different L-AMB formulations are influenced by the phospholipid type and the lipid/AMB ratio. The optimal ratio depends on the phospholipid itself. At a lipid/AMB ratio of 40, the antifungal activity depends mainly on the amount of unbound AMB in the formulation. At a lipid/AMB ratio of 1, the renal cell toxicity also depends on the fluidity of the phospholipid.     

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.     

Optimal use of existing and new antifungal drugs

Clinicians are increasingly aware that fungal pathogens are a significant cause of morbidity and mortality in hospitalized patients. Historically, these infections occurred in severely immunocompromised patients who were undergoing treatment for hematological malignancy or solid organ transplantation. Currently, however, systemic fungal infections are commonly seen in debilitated patients who are being nursed in intensive care or high-dependency units. These infections are mostly caused by Candida albicans but there is a growing proportion of strains of non- albicans Candida spp, some with reduced susceptibility to commonly used antifungals. The limited armamentarium of antifungal agents to date has meant that amphotericin B continues to be considered the most effective therapeutic agent albeit with a poor record of treatment-limiting side effects. The past decade has seen some encouraging developments in antifungal therapy. Three lipid formulations of amphotericin B showing reduced toxicity compared with the desoxycholate formulation are now licensed. There are three investigational triazoles currently undergoing evaluation that should prove important additions to existing members of this class. The echinocandin caspofungin is the first of a new class of antifungal agents with a novel mode of action, which has recently been approved for use in the United States.     

Comparison of in vitro antifungal activities of amphotericin B lipid complex with itraconazole against 708 clinical yeast isolates and opportunistic moulds determined by National Committee for Clinical Laboratory Standards methods M27-A and M38-P

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.     

Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms

Candida albicans is implicated in many biomaterial-related infections. Typically, these infections are associated with biofilm formation. Cells in biofilms display phenotypic traits that are dramatically different from those of their free-floating planktonic counterparts and are notoriously resistant to antimicrobial agents. Consequently, biofilm-related infections are inherently difficult to treat and to fully eradicate with normal treatment regimens. Here, we report a rapid and highly reproducible microtiter-based colorimetric assay for the susceptibility testing of fungal biofilms, based on the measurement of metabolic activities of the sessile cells by using a formazan salt reduction assay. The assay was used for in vitro antifungal susceptibility testing of several C. albicans strains grown as biofilms against amphotericin B and fluconazole and the increased resistance of C. albicans biofilms against these antifungal agents was demonstrated. Because of its simplicity, compatibility with a widely available 96-well microplate platform, high throughput, and automation potential, we believe this assay represents a promising tool for the standardization of in vitro antifungal susceptibility testing of fungal biofilms.     

Increased frequency of non-fumigatus Aspergillus species in amphotericin B- or triazole-pre-exposed cancer patients with positive cultures for aspergilli

Invasive aspergillosis (IA) can occur despite prior prophylactic or empiric use of triazoles or amphotericin B (AMB). Although profound immunosuppression may account for breakthrough IA, resistance of Aspergillus to antifungals may also play a role. To examine this question, we measured the minimal inhibitory concentration of 105 Aspergillus isolates recovered from 105 cancer patients (64 with IA, 41 with Aspergillus colonization) to AMB, itraconazole (ITC), and voriconazole (VRC) using the National Committee for Clinical Laboratory Standards (NCCLS) M38-A microdilution and E-test methods. We also determined the minimal fungicidal concentration (MFC) of these agents and the minimal effective concentration (MEC) of caspofungin (CAS) using standardized methods. We then collected information regarding pre-exposure to AMB or triazoles (fluconazole, ITC, VRC) within 3 months before Aspergillus isolation. Pre-exposure of cancer patients to AMB or triazoles was associated with increased frequency of non-fumigatus Aspergillus species. Aspergillus isolates recovered from patients who previously received AMB exhibited higher E-test AMB MICs compared with isolates from patients without prior AMB exposure (P = 0.01). In addition, the AMB MICs by E-test were higher in triazole-pre-exposed patients compared with those not exposed to triazoles (P = 0.001). The ITC and VRC MICs by E-test were not affected by prior AMB or triazole exposure. Finally, neither the AMB, ITC, and VRC MICs and MFCs by NCCLS method nor CAS MECs showed such changes. In conclusion, cancer patients with positive Aspergillus cultures who are pre-exposed to AMB or triazoles have high frequency of non-fumigatus Aspergillus species. These Aspergillus isolates were found to be AMB-resistant by the more sensitive E-test method.     

Antifungal combinations against Candida albicans biofilms in vitro

Candida biofilms display increased resistance to most antifungal agents. We have evaluated the efficacy of combinations of fluconazole (FLC), amphotericin B, and caspofungin (CSP) against Candida albicans biofilms in vitro. Indifference was observed for all the combinations of paired antifungal agents when a checkerboard titration method was used. Time-kill experiments revealed an antagonistic effect of high FLC doses with CSP.     

Comparative Susceptibility of Candida albicans to Amphotericin B and Amphotericin B Methyl Ester

The in vitro antifungal activities of amphotericin B (AMB) and amphotericin B methyl ester (AME) were compared against 465 clinical isolates of Candida albicans. AMB and AME possessed comparable activity against half of the strains, but against the remainder of the strains the activity of AME was slightly lower than that of AMB. Rarely did AME show superior antifungal activity to AMB.     

The value of amphotericin B in the treatment of invasive fungal infections

Over the last 20 years, there has been an increase in the total number of invasive fungal infections (IFIs) and in infections caused by rare and emerging pathogens. This is due in part to the growing population of immunocompromised patients at risk of developing fungal infections. Three classes of antifungal agents are widely used for the treatment of systemic fungal infections: polyenes, azoles, and echinocandins. Polyenes were the first antifungal agents developed and have a long-standing history in the treatment of IFIs. The use of conventional amphotericin B has been limited because of toxic side effects, which have been reduced by the lipid formulations of amphotericin B. Treatment options for invasive mycoses have expanded with the recent introduction of the second-generation triazoles (voriconazole and posaconazole) and the echinocandins (caspofungin, micafungin, anidulafungin). Despite the increased number of antifungal drugs, resistance issues present a problem in the treatment of IFIs. Although some fungal pathogens display innate resistance, others have developed resistance secondary to selective pressure. This article briefly reviews the changing epidemiology of fungal infections and associated risk factors, resistance issues with commonly administered antifungal agents, and treatment options for IFIs, with a focus on polyenes.     

Penetration of Candida biofilms by antifungal agents

A filter disk assay was used to investigate the penetration of antifungal agents through biofilms containing single and mixed-species biofilms containing Candida. Fluconazole permeated all single-species Candida biofilms more rapidly than flucytosine. The rates of diffusion of either drug through biofilms of three strains of Candida albicans were similar. However, the rates of drug diffusion through biofilms of C. glabrata or C. krusei were faster than those through biofilms of C. parapsilosis or C. tropicalis. In all cases, after 3 to 6 h the drug concentration at the distal edge of the biofilm was very high (many times the MIC). Nevertheless, drug penetration failed to produce complete killing of biofilm cells. These results indicate that poor antifungal penetration is not a major drug resistance mechanism for Candida biofilms. The abilities of flucytosine, fluconazole, amphotericin B, and voriconazole to penetrate mixed-species biofilms containing C. albicans and Staphylococcus epidermidis (a slime-producing wild-type strain, RP62A, and a slime-negative mutant, M7) were also investigated. All four antifungal agents diffused very slowly through these mixed-species biofilms. In most cases, diffusion was slower with biofilms containing S. epidermidis RP62A, but amphotericin B penetrated biofilms containing the M7 mutant more slowly. However, the drug concentrations reaching the distal edges of the biofilms always substantially exceeded the MIC. Thus, although the presence of bacteria and bacterial matrix material undoubtedly retarded the diffusion of the antifungal agents, poor penetration does not account for the drug resistance of Candida biofilm cells, even in these mixed-species biofilms.     

In vitro activity of 2-cyclohexylidenhydrazo-4-phenyl-thiazole compared with those of amphotericin B and fluconazole against clinical isolates of Candida spp. and fluconazole-resistant Candida albicans

OBJECTIVES: The aim of this study was to investigate the in vitro antifungal activity of an isothiosemicarbazone cyclic analogue against isolates of Candida spp. including fluconazole-resistant Candida albicans. METHODS: We investigated the activity of 2-cyclohexylidenhydrazo-4-phenyl-thiazole (EM-01D2) against 114 clinical isolates of Candida spp., representing five different species, by microdilution, according to the NCCLS method 27-A. The activity against C. albicans biofilms was also investigated. Toxicity in vitro was evaluated by MTT reduction assay. RESULTS: EM-01D2 demonstrated low toxicity, broad spectrum, fungicidal activity and was active against C. albicans and Candida krusei at concentrations lower than those shown by amphotericin B and fluconazole (P < 0.05). It maintained potent in vitro activity against fluconazole-resistant C. albicans isolates. Fungicidal activity occurred at concentrations 1-2 doubling dilutions greater than the corresponding MICs, and time-kill analysis indicated that a 99.9% loss of C. albicans viability occurred after 6 h of incubation in the presence of EM-01D2 at concentrations equal to four times the MIC. EM-01D2 was also active in inhibiting the growth of C. albicans ATCC 10231 biofilms, even though such inhibition occurred at concentrations higher than the MICs determined under planktonic growth conditions. However, when C. albicans biofilms were pre-exposed to subinhibitory concentrations of EM-01D2, a reduction of MIC50 of amphotericin B was observed. CONCLUSIONS: Based on these results, EM-01D2 could represent a template for the development of novel fungicidal agents.     

Absence of amphotericin B-tolerant persister cells in biofilms of some Candida species

Biofilms and planktonic cells of five Candida species were surveyed for the presence of persister (drug-tolerant) cell populations after exposure to amphotericin B. None of the planktonic cultures (exponential or stationary phase) contained persister cells. However, persisters were found in biofilms of one of two strains of Candida albicans tested and in biofilms of Candida krusei and Candida parapsilosis, but not in biofilms of Candida glabrata or Candida tropicalis. These results suggest that persister cells cannot solely account for drug resistance in Candida biofilms.     

Comparative analysis of amphotericin B lipid complex and liposomal amphotericin B kinetics of lung accumulation and fungal clearance in a murine model of acute invasive pulmonary aspergillosis

The reformulation of amphotericin B (AMB) into a lipid complex (AMB lipid complex [ABLC]) or liposomal carrier (liposomal AMB [L-AMB]) changes the rate and extent of drug distribution to the lung. The importance of pharmacokinetic differences among the various lipid AMB formulations in the treatment of invasive pulmonary aspergillosis (IPA) remains unknown. We compared the kinetics of AMB lung accumulation and fungal clearance of ABLC- and L-AMB-treated mice with acute IPA. BALB/c mice were immunosuppressed with cyclophosphamide and cortisone before intranasal inoculation with 1.5x10(6) Aspergillus fumigatus 293 conidia. ABLC or L-AMB was administered in daily intravenous doses (1, 5, or 10 mg/kg of body weight), starting 12 h after infection and continuing until day 5. At predetermined times (0, 24, 72, and 120 h), mice were euthanized, and lungs were harvested for determinations of lung fungal burdens (quantitative PCR) and total AMB lung tissue concentrations. Both ABLC and L-AMB were effective at reducing lung fungal burdens at doses of >or=5 mg/kg/day. Clearance of A. fumigatus during the first 24 h was associated with AMB tissue concentrations of >4 microg/g. At 5 mg/kg/day, ABLC produced a more rapid fungal clearance than did L-AMB, but at the end of therapy, fungal burden reductions were similar for both formulations and were not improved with higher dosages. These data suggest that ABLC delivers active AMB to the lung more rapidly than does L-AMB, resulting in faster Aspergillus clearance in an experimental model of IPA. However, pharmacodynamic differences between the two formulations were less apparent when mice were dosed at 10 mg/kg/day.     

Amphotericin B lipid complex therapy of experimental fungal infections in mice.

The amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, was evaluated for its acute toxicity in mice and for its efficacy in mice infected with a variety of fungal pathogens. ABLC was markedly less toxic to mice when it was administered intravenously; it had a 50% lethal dose of greater than 40 mg/kg compared with a 50% lethal dose of 3 mg/kg for Fungizone, the desoxycholate form of amphotericin B. ABLC was efficacious against systemic infections in mice caused by Candida albicans, Candida species other than C. albicans, Cryptococcus neoformans, and Histoplasma capsulatum. ABLC was also efficacious in immunocompromised animals infected with C. albicans, Aspergillus fumigatus, and H. capsulatum. Against some infections, the efficacy of ABLC was comparable to that of Fungizone, while against other infections Fungizone was two- to fourfold more effective than ABLC. Against several infections. Fungizone could not be given at therapeutic levels because of intravenous toxicity. ABLC, with its reduced toxicity, could be administered at drug levels capable of giving a therapeutic response. ABLC should be of value in the treatment of severe fungal infections in humans.     

Susceptibility of Cryptococcus neoformans biofilms to antifungal agents in vitro

Microbial biofilms contribute to virulence and resistance to antibiotics by shielding microbial cells from host defenses and antimicrobial drugs, respectively. Cryptococcus neoformans was demonstrated to form biofilms in polystyrene microtiter plates. The numbers of CFU of disaggregated biofilms, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide reduction, and light and confocal microscopy were used to measure the fungal mass, the metabolic activity, and the appearance of C. neoformans biofilms, respectively. Biofilm development by C. neoformans followed a standard sequence of events: fungal surface attachment, microcolony formation, and matrix production. The susceptibilities of C. neoformans cells of the biofilm and planktonic phenotypes to four antifungal agents were examined. The exposure of C. neoformans cells or preformed cryptococcal biofilms to fluconazole or voriconazole did not result in yeast growth inhibition and did not affect the metabolic activities of the biofilms, respectively. In contrast, both C. neoformans cells and preformed biofilms were susceptible to amphotericin B and caspofungin. However, C. neoformans biofilms were significantly more resistant to amphotericin B and caspofungin than planktonic cells, and their susceptibilities to these drugs were further reduced if cryptococcal cells contained melanin. A spot enzyme-linked immunosorbent assay and light and confocal microscopy were used to investigate how antifungal drugs affected C. neoformans biofilm formation. The mechanism by which amphotericin B and caspofungin interfered with C. neoformans biofilm formation involved capsular polysaccharide release and adherence. Our results suggest that biofilm formation may diminish the efficacies of some antifungal drugs during cryptococcal infection.