In Vitro Activity of a New Polyene, SPA-S-843, against Yeasts

The in vitro activity of a new water-soluble polyene, SPA-S-843, was evaluated against 116 strains of Candida, Cryptococcus, and Saccharomyces spp. and compared with that of amphotericin B. SPA-S-843 demonstrated better inhibitory activity against all of the yeasts examined and better fungicidal activity against Candida albicans, Candida glabrata, Candida krusei, and Candida tropicalis than did amphotericin B.     

In Vitro Antifungal Activities of a Series of Dication-Substituted Carbazoles,Furans, and Benzimidazoles

Aromatic dicationic compounds possess antimicrobial activity against a wide range of eucaryotic pathogens, and in the present study an examination of the structures-functions of a series of compounds against fungi was performed. Sixty-seven dicationic molecules were screened for their inhibitory and fungicidal activities against Candida albicans and Cryptococcus neoformans. The MICs of a large number of compounds were comparable to those of the standard antifungal drugs amphotericin B and fluconazole. Unlike fluconazole, potent inhibitory compounds in this series were found to have excellent fungicidal activities. The MIC of one of the most potent compounds against C. albicans was 0.39 μg/ml, and it was the most potent compound against C. neoformans (MIC, ≤0.09 μg/ml). Selected compounds were also found to be active against Aspergillus fumigatus, Fusarium solani, Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans. Since some of these compounds have been safely given to animals, these classes of molecules have the potential to be developed as antifungal agents.The incidence of fungal infections in the immunocompromised population has significantly increased over the past two decades. Frequent infections caused by molds which may be primarily resistant to azoles and azole-resistant isolates of Candida albicans and Cryptococcus neoformans, which have developed recently, have increasingly been reported (7, 13, 23, 30). In light of these developments, new antifungal agents with various mechanisms of action and fungicidal activities are needed for the effective management of these clinically important infections.Recently, we reported on the antifungal activities of analogues and metabolites of pentamidine and a series of dicationic substituted bis-benzimidazoles (11). Those in vitro studies uncovered a number of compounds with potent activity against C. albicans and C. neoformans. Several compounds were found to have inhibitory activity against these two fungi more potent than that of either fluconazole or amphotericin B. In addition, the dicationic molecules, unlike fluconazole, proved to have potent fungicidal activity, with the most potent compounds having minimum fungicidal concentrations (MFCs) below 1.0 μg/ml. These initial studies also found that dicationic molecules were effective against Aspergillus fumigatus, Fusarium solani, several Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans.On the basis of the initial promising results reported above, the current work expands our studies on the antifungal activities of dication-substituted molecules by screening 67 additional compounds against C. albicans and C. neoformans. The criteria used to choose the structures for the current studies were based on years of testing dicationic molecules against the fungus Pneumocystis carinii in a rat model of disease (5, 14, 17, 18, 24, 26–28). Compounds in the current studies include molecules with the cationic moieties linked by carbazole, furan, and benzimidazole bridges. In addition to screening all compounds against C. albicans and C. neoformans, selected compounds were tested against other yeasts, molds, and azole-resistant strains of C. albicans and C. neoformans.     

Superiority of Amphotericin over Nystatin in Thayer-Martin Medium

Vaginal and cervical specimens taken for the detection of gonorrhea frequently contain Candida albicans. Thayer-Martin medium containing nystatin (VCTN) is unsatisfactory because nystatin fails to selectively inhibit C. albicans. Thayer-Martin medium containing no nystatin was prepared with varying concentrations of amphotericin (VCTA) added, and this latter medium was compared with VCTN for its ability to selectively inhibit varying numbers of C. albicans. The effect of amphotericin on the recovery of varying numbers of Neisseria gonorrhoeae was also studied. VCTN failed to inhibit 10¹ to 10⁶C. albicans. VCTA suppressed growth of up to 10⁶C. albicans. The optimal concentration of amphotericin B appeared to be 100 μg/ml. VCTA containing 100 μg of amphotericin B per ml (VCTA 100) was not inhibitory for N. gonorrhoeae when a dense population was applied with a replicator. However, it was slightly inhibitory when 10¹ to 10²N. gonorrhoeae were applied over the entire surface of an agar plate, although the colony count on VCTA 100 was more than 50% of the counts on chocolate agar and 75% of the counts on VCTN. These properties of VCTA 100, when stored at 4 C, were stable for up to 2 weeks.     

Synergistic Activity of the Tyrocidines,Antimicrobial Cyclodecapeptides from Bacillus aneurinolyticus,with Amphotericin B and Caspofungin against Candida albicans Biofilms

Tyrocidines are cationic cyclodecapeptides from Bacillus aneurinolyticus that are characterized by potent antibacterial and antimalarial activities. In this study, we show that various tyrocidines have significant activity against planktonic Candida albicans in the low-micromolar range. These tyrocidines also prevented C. albicans biofilm formation in vitro. Studies with the membrane-impermeable dye propidium iodide showed that the tyrocidines disrupt the membrane integrity of mature C. albicans biofilm cells. This membrane activity correlated with the permeabilization and rapid lysis of model fungal membranes containing phosphatidylcholine and ergosterol (70:30 ratio) induced by the tyrocidines. The tyrocidines exhibited pronounced synergistic biofilm-eradicating activity in combination with two key antifungal drugs, amphotericin B and caspofungin. Using a Caenorhabditis elegans infection model, we found that tyrocidine A potentiated the activity of caspofungin. Therefore, tyrocidines are promising candidates for further research as antifungal drugs and as agents for combinatorial treatment.     

Efficacies of KY62 against Leishmania amazonensis and Leishmania donovani in Experimental Murine Cutaneous Leishmaniasis and Visceral Leishmaniasis

Current therapy for leishmaniasis is unsatisfactory because parenteral antimonial salts and pentamidine are associated with significant toxicity and failure rates. We examined the efficacy of KY62, a new, water-soluble, polyene antifungal, against cutaneous infection with Leishmania amazonensis and against visceral infection with Leishmania donovani in susceptible BALB/c mice. Mice were infected with L. amazonensis promastigotes in the ear pinna and in the tail and were treated with KY62 or amphotericin B. The cutaneous lesions showed a remarkable response to therapy with KY62 at a dose of 30 mg per kg of body weight per day. At this dose, the efficacy of KY62 was equivalent to or better than that of amphotericin B at 1 to 5 mg/kg/day. Mice infected intravenously with 10⁷ L. donovani promastigotes and treated with KY62 showed a 4-log reduction in the parasite burden in the liver and spleen compared to untreated mice. These studies indicate potent activity of KY62 against experimental cutaneous leishmaniasis caused by L. amazoniensis and against experimental visceral leishmaniasis caused by L. donovani.     

Effect of aqueous extract of date palm fruit (<Emphasis Type="Italic">Phoenix dactylifera</Emphasis> L.) on therapeutic index of amphotericin B

The aim of this study was to identify the effect of aqueous extract of date palm fruit (Phoenix dactylifera L.) on therapeutic index of amphotericin B. The water extract of date fruit of Phoenix dactylifera were tested for the antifungal activity with amphotericin B against the yeast Candida albicans ATCC 1023. Secondly, we have tested the cytotoxicity of this complex on human red blood cells.The results showed a significant improvement in the antifungal activity compared with amphotericin B alone at therapeutic concentration. Furthermore, the addition of aqueous extract protects human red blood cells against the cytotoxicity induced by amphotericin B. This is due probably, to the effect of flavonoïds and polysaccharides present in date fruit.     

Experimental Urinary Tract Infection in Rats Caused by Candida albicans

The increased clinical use of broad-spectrum antibiotics, immunosuppressives, and anti-inflammatory agents is thought to have contributed to the increased incidence of renal infections due to Candida albicans. To aid in the search for improved anticandidal agents, we developed an experimental model in which C. albicans is injected into the medulla of the surgically exposed kidney of the rat. The ensuing infection is restricted to the urinary tract and can be treated successfully with amphotericin B. This model promises to be of merit in the evaluation of agents potentially useful in treating candidiasis of the urinary tract.     

Assessment of Antifungal Activities of Fluconazole and Amphotericin B Administered Alone and in Combination against Candida albicans by Using a Dynamic In Vitro Mycotic Infection Model

We evaluated the pharmacodynamic activities of fluconazole and amphotericin B given alone and in combination against Candida albicans by using an in vitro model of bloodstream infection that simulates human serum pharmacokinetic parameters for these antifungals. Fluconazole was administered as a bolus into the model to simulate regimens of 200 mg every 24 h (q24h) and 400 mg q24h. Amphotericin B was administered at doses producing the peak concentration (2.4 μg/ml) observed with a regimen of 1 mg/kg of body weight q24h. A combination regimen of fluconazole (400 mg q24h) and amphotericin B (1 mg/kg q24h) administered simultaneously and as a staggered regimen (amphotericin B bolus given 8 h after fluconazole bolus) was also simulated in the model to characterize possible antagonism between these agents. Fluconazole alone and amphotericin B alone demonstrated fungistatic (<99.9% reduction in numbers of CFU per milliliter from the starting inoculum) and fungicidal (>99.9% reduction) activity, respectively. When fluconazole and amphotericin B were administered simultaneously, fungicidal activity similar to that observed with amphotericin B alone was observed. Staggered administration of fluconazole and amphotericin B, however, resulted in a substantial reduction of the fungicidal activity of amphotericin B, producing fungistatic activity similar to that observed with noncombination fluconazole regimens. These results demonstrate the usefulness of this model for comparing the in vitro pharmacodynamic characteristics of different antifungal regimens and support the theory of azole-polyene antagonism. The effects of this antagonism on the in vivo activity and clinical usefulness of combination antifungal therapy, however, remain to be determined.     

Superior Efficacy of Liposomal Amphotericin B with Prolonged Circulation in Blood in the Treatment of Severe Candidiasis in Leukopenic Mice

In leukopenic mice with severe systemic candidiasis, single-dose treatment (5 mg of amphotericin B [AMB]/kg of body weight) with long-circulating polyethylene glycol-coated AMB liposomes (PEG-AMB-LIP) resulted in zero mortality and a significant reduction in the number of viable Candida albicans in the kidney, whereas 70% mortality was seen in mice treated with five daily doses of AmBisome (5 mg of AMB/kg · day). When the first of five daily doses of AmBisome was combined with a single low dose of Fungizone (0.1 mg of AMB/kg), the efficacy was equal to that of PEG-AMB-LIP.     

Amphotericin-Associated Infusion-Related Reactions: A Narrative Review of Pre-Medications

PurposeAmphotericin B has been reported to cause infusion-related adverse effects (IRAEs). To prevent IRAEs, pre-medications may be administered prior to the administration of amphotericin B. The effects of different formulations of amphotericin B (amphotericin B deoxycholate and lipid formulations), duration of infusion, and utility of pre-medications in preventing IRAEs are reviewed.MethodsPubMed, Ovid Medline, Embase, Web of Science, the Cochrane Database of Systematic Reviews, the Cochrane Central Register of Controlled Trials, and the Scopus databases were searched with the following search terms: pre-medication, amphotericin B, and its related compounds. Upon review, a total of 39 publications were considered for inclusion.FindingsIn vitro and in vivo studies have reported that amphotericin B deoxycholate stimulates pro-inflammatory cytokine genes causing IRAEs. Nonetheless, the clinical literature has reported that IRAEs occur among patients who received pre-medications. In comparison to amphotericin B deoxycholate, lipid-based formulations of amphotericin may result in a lower or similar risk for IRAEs.ImplicationsThe routine use of pre-medications to prevent IRAEs after the administration of amphotericin B (amphotericin B deoxycholate or lipid formulations) would not be warranted.     

Radiometric Determination of the Concentration of Amphotericin B in Body Fluids

A new assay was developed to determine the concentration of amphotericin B in body fluids. The Bactec radiometric system was used to measure CO₂ production by a test strain, Candida albicans MDA 448, in the presence of amphotericin B. After 5-h incubation, drug concentrations as low as 0.2 μg/ml could be detected. The results are comparable to those of the commonly used agar diffusion assay with Paecilomyces varioti.     

Efficacy of LY303366 against Amphotericin B-Susceptible and -Resistant Aspergillus fumigatus in a Murine Model of Invasive Aspergillosis

LY303366 is a novel antifungal echinocandin with excellent in vitro activity against Aspergillus spp. We compared four doses (1, 2.5, 10, and 25 mg/kg of body weight) of LY303366 with amphotericin B (0.5 to 5 mg/kg) in a temporarily neutropenic murine model of invasive aspergillosis against an amphotericin B-susceptible (AF210) and an amphotericin B-resistant (AF65) Aspergillus fumigatus isolate based on in vivo response. Mice were immunosuppressed with cyclophosphamide (200 mg/kg) and infected 3 days later. Treatment started 18 h after infection and lasted for 10 days. LY303366 was given once daily intravenously for 10 days, and amphotericin B (at 0.5, 2, and 5 mg/kg) was given once daily intraperitoneally for 10 days, or only on days 1, 2, 4, and 7 (at 5 mg/kg). Kidneys and lungs from survivors were cultured on day 11. Control mice in both experiments had 90 to 100% mortality. Amphotericin B at 0.5 mg/kg and LY303366 at 1 mg/kg yielded 10 to 20% survival rates for mice infected with either AF210 or AF65. Amphotericin B at 2 and 5 (both regimens) mg/kg yielded a 70 to 100% survival rate for mice infected with AF210 but a 10 to 30% survival rate for mice infected with AF65 (P = 0.01 to 0.04 compared with AF210). Against AF210 and AF65, LY303366 at 2.5, 10, and 25 mg/kg produced a survival rate of 70 to 80%, which was as effective as amphotericin B for AF210, but superior to amphotericin B for AF65 (P < 0.03 to 0.0006). For AF65, LY303366 at 10 and 25 mg/kg/day was superior to amphotericin B at 2 and 5 mg/kg/day in reducing tissue colony counts (P = 0.01 to 0.003), and for AF210, amphotericin B at 5 mg/kg/day and at 5 mg/kg in four doses was more effective than all four regimens of LY303366 in reducing renal culture counts (P = 0.01 to 0.0001). The present study shows, for the first time, that in vivo resistance of A. fumigatus to amphotericin B exists, although this could not be detected by in vitro susceptibility assays. Furthermore, LY303366 appears to be effective against amphotericin B-susceptible and -resistant A. fumigatus infection in this model and should be further evaluated clinically.     

Therapeutic effect of the triazole Bay R 3783 in mouse models of coccidioidomycosis, blastomycosis, and histoplasmosis.

A new triazole, Bay R 3783, was compared with ketoconazole, itraconazole, and fluconazole, which were given via the alimentary tract at three dosages, and amphotericin B, which was given at 1 mg/kg intraperitoneally, in murine models of the systemic mycoses coccidioidomycosis, histoplasmosis, and blastomycosis. In a pulmonary coccidioidomycosis model, Bay R 3783, fluconazole, and itraconazole were essentially equally efficacious and more active than ketoconazole in protecting mice against death; but they were inferior to amphotericin B. In a short-term organ load experiment, Bay R 3783 and amphotericin B were equally effective and were more effective than the other drugs in reducing the amount of Coccidioides immitis in the lungs. Against meningocerebral coccidioidomycosis, Bay R 3783, itraconazole, and fluconazole at 25 mg/kg and amphotericin B prevented death only during therapy, with mortalities ensuing shortly thereafter. In mice with systemic histoplasmosis, Bay R 3783 and itraconazole at 25 mg/kg and amphotericin B prevented death in all mice through a 44-day observation period. Clearance of Histoplasma capsulatum from organs was similar in mice treated with Bay R 3783 and itraconazole; this clearance was greater than that in mice treated with ketoconazole and fluconazole but less than that in mice treated with amphotericin B. In mice with systemic blastomycosis, Bay R 3783 at 25 mg/kg yielded 90% survivors at 60 days, which was greater than that achieved with amphotericin B (60%) or itraconazole (30%). Clearance of Blastomyces dermatitidis from the lungs was greatest with Bay R 3783, followed by that with amphotericin B, itraconazole, fluconazole, and ketoconazole, in that order. Therefore, Bay R 3783 showed effectiveness comparable to or exceeding those of itraconazole and fluconazole and exceeding that of ketoconazole against these systemic mycoses in mice.     

Activity of Liposomal Amphotericin B with Prolonged Circulation in Blood versus Those of AmBisome and Fungizone against Intracellular Candida albicans in Murine Peritoneal Macrophages

Activity against intracellular Candida albicans was assessed in C. albicans-infected murine peritoneal macrophages exposed to long-circulating pegylated amphotericin B liposomes (PEG-AMB-LIP), AmBisome, or Fungizone. The level of antifungal activity of Fungizone is much higher than that of AmBisome or PEG-AMB-LIP, while PEG-AMB-LIP and AmBisome show equivalent activity levels. Previous exposure of uninfected macrophages to PEG-AMB-LIP or AmBisome is advantageous for intracellular antifungal activity.     

Artemisinins,New Miconazole Potentiators Resulting in Increased Activity against Candida albicans Biofilms

Mucosal biofilm-related fungal infections are very common, and the incidence of recurrent oral and vulvovaginal candidiasis is significant. As resistance to azoles (the preferred treatment) is occurring, we aimed at identifying compounds that increase the activity of miconazole against Candida albicans biofilms. We screened 1,600 compounds of a drug-repositioning library in combination with a subinhibitory concentration of miconazole. Synergy between the best identified potentiators and miconazole was characterized by checkerboard analyses and fractional inhibitory concentration indices. Hexachlorophene, pyrvinium pamoate, and artesunate act synergistically with miconazole in affecting C. albicans biofilms. Synergy was most pronounced for artesunate and structural homologues thereof. No synergistic effect could be observed between artesunate and fluconazole, caspofungin, or amphotericin B. Our data reveal enhancement of the antibiofilm activity of miconazole by artesunate, pointing to potential combination therapy consisting of miconazole and artesunate to treat C. albicans biofilm-related infections.     

Effect of Growth Rate on Resistance of Candida albicans Biofilms to Antifungal Agents

A perfused biofilm fermentor, which allows growth-rate control of adherent microbial populations, was used to assess whether the susceptibility of Candida albicans biofilms to antifungal agents is dependent on growth rate. Biofilms were generated under conditions of glucose limitation and were perfused with drugs at a high concentration (20 times the MIC). Amphotericin B produced a greater reduction in the number of daughter cells in biofilm eluates than ketoconazole, fluconazole, or flucytosine. Similar decreases in daughter cell counts were observed when biofilms growing at three different rates were perfused with amphotericin B. In a separate series of experiments, intact biofilms, resuspended biofilm cells, and newly formed daughter cells were removed from the fermentor and were exposed to a lower concentration of amphotericin B for 1 h. The susceptibility profiles over a range of growth rates were then compared with those obtained for planktonic cells grown at the same rates under glucose limitation in a chemostat. Intact biofilms were resistant to amphotericin B at all growth rates tested, whereas planktonic cells were resistant only at low growth rates (≤0.13 h⁻¹). Cells resuspended from biofilms were less resistant than intact biofilm populations but more resistant than daughter cells; the susceptibilities of both these cell types were largely independent of growth rate. Our findings indicate that the amphotericin B resistance of C. albicans biofilms is not simply due to a low growth rate but depends on some other feature of the biofilm mode of growth.     

Inhibition of Nucleic Acid Biosynthesis Makes Little Difference to Formation of Amphotericin B-Tolerant Persisters in Candida albicans Biofilm

Candida albicans persisters constitute a small subpopulation of biofilm cells and play a major role in recalcitrant chronic candidiasis; however, the mechanism underlying persister formation remains unclear. Persisters are often described as dormant, multidrug-tolerant, nongrowing cells. Persister cells are difficult to isolate and study not only due to their low levels in C. albicans biofilms but also due to their transient, reversible phenotype. In this study, we tried to induce persister formation by inducing C. albicans cells into a dormant state. C. albicans cells were pretreated with 5-fluorocytosine (planktonic cells, 0.8 μg ml⁻¹; biofilm cells, 1 μg ml⁻¹) for 6 h at 37°C, which inhibits nucleic acid and protein synthesis. Biofilms and planktonic cultures of eight C. albicans strains were surveyed for persisters after amphotericin B treatment (100 μg ml⁻¹ for 24 h) and CFU assay. None of the planktonic cultures, with or without 5-fluorocytosine pretreatment, contained persisters. Persister cells were found in biofilms of all tested C. albicans strains, representing approximately 0.01 to 1.93% of the total population. However, the persister levels were not significantly increased in C. albicans biofilms pretreated with 5-fluorocytosine. These results suggest that inhibition of nucleic acid synthesis did not seem to increase the formation of amphotericin B-tolerant persisters in C. albicans biofilms.     

évaluation de l’activité antifongique de différents extraits de la cannelle de Chine (Cinnamomum cassia)

The incidence of invasive fungal infections due to Candida albicans has dramatically increased since 25 years. The amphotericin B remains the best treatment despite its severe toxicity. Our work is inscribed in the frame of finding of new natural antifungals agents from a condiment widely used in our diet: the Chinese cinnamon (Cinnamomum cassia). This study is focused on the qualitative determination of different families of secondary metabolites from the bark of Cinnamon. It is also focused on assessing the antifungal activity of some extracts of Cinnamon. The plant material was extracted by exhaustion using increased polarity solvents (chloroform, ethyl acetate, methanol and water). We made five exhaustions for each solvent, each one was tested separately. The phytochemical study revealed the presence of terpenes, alkaloids and polyphenols mainly represented by flavonoids. Evaluation of antimicrobial activity of the various extracts was carried out against references yeasts strains Candida albicans ATCC 10231 and Candida albicans 444IP. Results showed that in the exception of aqueous extracts, all other extracts have an interesting activity, with inhibition zone diameters between 19 and 60 mm for the chloroform extract. Similar results were obtained for the other organic extracts. Indeed, extracts obtained from low or medium polarity solvents are the most active. In addition, the MIC and MFC of the first fraction of the chloroform extract were respectively 0.10 and 0.20 μg/ml against Candida albicans ATCC 10231. They remain lower than those of amphotericin B against the same strain (MIC = 0.2 μg/ml and MFC = 0.4 μg/ml).     

In Vitro Analysis of Finasteride Activity against Candida albicans Urinary Biofilm Formation and Filamentation

Candida albicans is the 3rd most common cause of catheter-associated urinary tract infections, with a strong propensity to form drug-resistant catheter-related biofilms. Due to the limited efficacy of available antifungals against biofilms, drug repurposing has been investigated in order to identify novel agents with activities against fungal biofilms. Finasteride is a 5-α-reductase inhibitor commonly used for the treatment of benign prostatic hyperplasia, with activity against human type II and III isoenzymes. We analyzed the Candida Genome Database and identified a C. albicans homolog of type III 5-α-reductase, Dfg10p, which shares 27% sequence identity and 41% similarity to the human type III 5-α-reductase. Thus, we investigated finasteride for activity against C. albicans urinary biofilms, alone and in combination with amphotericin B or fluconazole. Finasteride alone was highly effective in the prevention of C. albicans biofilm formation at doses of ≥16 mg/liter and the treatment of preformed biofilms at doses of ≥128 mg/liter. In biofilm checkerboard analyses, finasteride exhibited synergistic activity in the prevention of biofilm formation in a combination of 4 mg/liter finasteride with 2 mg/liter fluconazole. Finasteride inhibited filamentation, thus suggesting a potential mechanism of action. These results indicate that finasteride alone is highly active in the prevention of C. albicans urinary biofilms in vitro and has synergistic activity in combination with fluconazole. Further investigation of the clinical utility of finasteride in the prevention of urinary candidiasis is warranted.     

Activities of Fluconazole,Caspofungin, Anidulafungin,and Amphotericin B on Planktonic and Biofilm Candida Species Determined by Microcalorimetry

We investigated the activities of fluconazole, caspofungin, anidulafungin, and amphotericin B against Candida species in planktonic form and biofilms using a highly sensitive assay measuring growth-related heat production (microcalorimetry). C. albicans, C. glabrata, C. krusei, and C. parapsilosis were tested, and MICs were determined by the broth microdilution method. The antifungal activities were determined by isothermal microcalorimetry at 37°C in RPMI 1640. For planktonic Candida, heat flow was measured in the presence of antifungal dilutions for 24 h. Candida biofilm was formed on porous glass beads for 24 h and exposed to serial dilutions of antifungals for 24 h, and heat flow was measured for 48 h. The minimum heat inhibitory concentration (MHIC) was defined as the lowest antifungal concentration reducing the heat flow peak by ≥50% (≥90% for amphotericin B) at 24 h for planktonic Candida and at 48 h for Candida biofilms (measured also at 24 h). Fluconazole (planktonic MHICs, 0.25 to >512 μg/ml) and amphotericin B (planktonic MHICs, 0.25 to 1 μg/ml) showed higher MHICs than anidulafungin (planktonic MHICs, 0.015 to 0.5 μg/ml) and caspofungin (planktonic MHICs, 0.125 to 0.5 μg/ml). Against Candida species in biofilms, fluconazole''s activity was reduced by >1,000-fold compared to its activity against the planktonic counterparts, whereas echinocandins and amphotericin B mainly preserved their activities. Fluconazole induced growth of planktonic C. krusei at sub-MICs. At high concentrations of caspofungin (>4 μg/ml), paradoxical growth of planktonic C. albicans and C. glabrata was observed. Microcalorimetry enabled real-time evaluation of antifungal activities against planktonic and biofilm Candida organisms. It can be used in the future to evaluate new antifungals and antifungal combinations and to study resistant strains.