Effects of amphotericin B on Aspergillus flavus clinical isolates with variable susceptibilities to the polyene in an experimental model of systemic aspergillosis.

OBJECTIVES: The aim of the present study was to evaluate the effects of amphotericin B (AMB) on clinical isolates of Aspergillus flavus. METHODS: MICs of both standard AMB and liposomal AMB (L-AMB) were determined using a broth dilution method for seven isolates of A. flavus. AMB MICs were also determined using the Etest. The activity of the polyene was then investigated in a murine model of systemic aspergillosis in which animals were infected intravenously, treated intravenously with several doses of the polyene (1-10 mg/kg/day) and observed for survival. RESULTS: Broth dilution AMB, broth dilution L-AMB and Etest AMB MICs ranged from 0.5 to 2.0 mg/L, 0.06 to >16 mg/L and 1.0 to >32 mg/L, respectively. There were two isolates for which all doses were effective at prolonging the survival. Their AMB MICs were ≤1.0 mg/L, regardless of the method/drug formulation utilized for testing. There were four isolates for which no regimen was effective. Their broth dilution AMB, broth dilution L-AMB and Etest AMB MICs ranged from 1.0 to 2.0 mg/L, 0.06 to >16 mg/L and 2.0 to >32 mg/L, respectively. There was one isolate for which only L-AMB given at 10 mg/kg/day was effective; broth dilution MICs of AMB and L-AMB were 0.5 mg/L, while the Etest MIC of AMB was 2.0 mg/L. CONCLUSIONS: Our data indicate that not all isolates of A. flavus should be considered resistant to AMB. The Etest represented the in vitro method that best correlated with the experimental infection. Finally, a clinical isolate showing an MIC ≥2.0 mg/L may be reasonably considered resistant in vivo to any dose/formulation of the polyene.

In vitro and in vivo effects of echinocandins against Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis.

OBJECTIVES: The aim of the present study was to compare, in vitro and in vivo, the effects of caspofungin, micafungin and anidulafungin against Candida parapsilosis complex isolates. METHODS: In vitro activities of all three echinocandins were assessed against C. parapsilosis sensu stricto (n = 4), Candida orthopsilosis (n = 4) and Candida metapsilosis (n = 3) using broth microdilution susceptibility testing, minimum fungicidal concentration determination and a killing-curve assay, in the absence and in the presence of 50% human serum. Then, the activities of all drugs were investigated in an immunocompromised murine model of systemic candidiasis. Animals were infected with six isolates (two for each species) and treated with the echinocandins administered at 0.25, 1, 5 and 10 mg/kg/day for six consecutive days. Fungal burdens were assessed in kidney tissues on day 7 post-infection. RESULTS: Geometric mean MICs of caspofungin, micafungin and anidulafungin for C. parapsilosis sensu lato were, respectively, 0.09, 0.14 and 0.20 mg/L without serum, and 0.70, 3.92 and 5.84 mg/L with serum. The fungicidal activity of all three echinocandins was variable; however, the addition of serum reduced the fungicidal effects against these species. In vivo studies showed that caspofungin at 5 and 10 mg/kg/day significantly decreased the kidney burdens with respect to the controls for all isolates, while micafungin was active at 5 and/or 10 mg/kg/day only against C. metapsilosis. CONCLUSIONS: Our susceptibility testing showed that caspofungin was the most active echinocandin against all three species. Also, caspofungin resulted in significant therapeutic effects for treatments of experimental systemic infections due to the three species, while micafungin was effective only against C. metapsilosis.

Comparative effects of micafungin, caspofungin, and anidulafungin against a difficult-to-treat fungal opportunistic pathogen, Candida glabrata.

The aim of this study was to compare the in vitro and in vivo activities of micafungin, caspofungin, and anidulafungin against Candida glabrata. The MICs against 28 clinical isolates showed that the overall susceptibilities to caspofungin and to micafungin were not statistically different in the absence of human serum, whereas the isolates were less susceptible to micafungin than to caspofungin in its presence. Minimum fungicidal concentrations, as well as time-kill experiments, showed that caspofungin was more active than anidulafungin, while micafungin was superior to either caspofungin or anidulafungin without serum; its addition rendered caspofungin and micafungin equally effective. A murine model of systemic candidiasis against a C. glabrata-susceptible isolate was performed to study the effects of all three echinocandins, and kidney burden counts showed that caspofungin, micafungin, and anidulafungin were active starting from 0.25, 1, and 5 mg/kg of body weight/day, respectively. Two echinocandin-resistant strains of C. glabrata were selected: C. glabrata 30, a laboratory strain harboring the mutation Fks2p-P667T, and C. glabrata 51, a clinical isolate harboring the mutation Fks2p-D666G. Micafungin activity was shown to be as effective as or more effective than that of caspofungin or anidulafungin in terms of MICs. In vivo studies against these resistant strains showed that micafungin was active starting from 1 mg/kg/day, while caspofungin was effective only when administrated at higher doses of 5 or 10 mg/kg/day. Although a trend toward colony reduction was observed with the highest doses of anidulafungin, a significant statistical difference was never reached.

In vitro and in vivo activities of posaconazole against zygomycetes with various degrees of susceptibility.

OBJECTIVES: We analysed the in vitro and in vivo effects of posaconazole and amphotericin B against three clinical isolates of zygomycetes: Lichtheimia corymbifera, F1; and Rhizopus oryzae, F5 and F6. METHODS: In vitro activities of both drugs were assessed by determining MICs, minimum fungicidal concentrations (MFCs) and fungal damage measured by the XTT assay against either the spores or the hyphal forms. Additionally, the survival curves of neutropenic mice systemically infected with the zygomycete isolates were used as the marker of antifungal response to amphotericin B (1 mg/kg/day) or posaconazole (2.5, 10 and 50 mg/kg/day). RESULTS: In terms of MICs, posaconazole proved to be active against the three isolates (MICs ranging from 0.125 to 1.0 mg/L). The median posaconazole MFCs were 0.25, 0.5 and >16 mg/L for F1, F5 and F6, respectively. The XTT assay showed that posaconazole was active against spores of all three isolates, but only partially effective against the hyphae. The survival studies showed that amphotericin B at 1 mg/kg/day and posaconazole at 10 mg/kg/day prolonged the survival of the animals infected with L. corymbifera F1. In mice infected with R. oryzae F5, only posaconazole at 50 mg/kg/day significantly prolonged survival, whereas amphotericin B at 1 mg/kg/day was the only regimen active against R. oryzae F6. CONCLUSIONS: Our findings showed that posaconazole could be useful in the treatment of zygomycosis. Also, we report that an isolate of R. oryzae with low MFC responded to posaconazole, while another isolate with high MFC did not.

Disruption of homocitrate synthase genes in Candida albicans affects growth but not virulence.

Two genes, LYS21 and LYS22, encoding isoforms of homocitrate synthase, an enzyme catalysing the first committed step in the lysine biosynthetic pathway, were disrupted in Candida albicans using the SAT1 flipper strategy. The double null lys21Δ/lys22Δ mutant lacked homocitrate synthase activity and exhibited lysine auxotrophy in minimal media that could be fully rescued by the addition of 0.5-0.6 mM L: -lysine. On the other hand, its virulence in vivo in the model of disseminated murine candidiasis appeared identical to that of the mother, wild-type strain. These findings strongly question a possibility of exploitation of homocitrate synthase and possibly also other enzymes of the lysine biosynthetic pathway as targets in chemotherapy of disseminated fungal infections.

Anidulafungin in combination with amphotericin B against Aspergillus fumigatus.

We investigated the effects of anidulafungin alone and in combination with amphotericin B against Aspergillus fumigatus. Indifference was the only type of interaction observed in vitro. Anidulafungin at 1 and 5 mg/kg of body weight/day, amphotericin B at 1 mg/kg/day, and combination therapy prolonged the survival of mice with invasive aspergillosis. Anidulafungin at 5 mg/kg/day, alone and in combination with amphotericin B, reduced the kidney fungal burden. Overall, the combination was not superior to the most active single drug.

In vitro susceptibility of dermatophytes to conventional and alternative antifungal agents.

The minimum inhibitory concentrations (MICs), the minimal fungicidal concentrations (MFCs), the fungal biomass (FB) and hyphal viability employing the dye 3-4,5 dimethyl- 2-thiazolyl- 2,5- diphenyl- 2H tetrazolium bromide (MTT) were used to compare the in vitro effects of fluconazole (FLU) with those of the N-terminal palmitoyl-lipidated peptide, Pal-Lys-Lys-NH(2) (PAL), and a tea tree oil component, gamma-Terpinene (TER), against several clinical isolates of Microsporum canis and Trichophyton rubrum. In general, FLU and PAL MICs were significantly lower than those observed with TER, while no differences in the three drugs were found in the MFCs. However, they were from two to 16-times higher than their respective MICs. FB of M. canis treated with either FLU or PAL, but not with TER, was significantly reduced over untreated controls. Only PAL and TER, in a medium-dependent fashion, but not FLU, reduced the FB of T. rubrum. Finally, PAL was found to be significantly more active than FLU at reducing the hyphal viability against both genera of dermatophytes. This study shows that PAL exerts an in vitro activity against dermatophytes at least similar to that observed with FLU and suggests that this compound might be a promising candidate in the treatment of infections due to dermatophytes.

Evaluation of the disk diffusion method compared to the microdilution method in susceptibility testing of anidulafungin against filamentous fungi.

Susceptibility testing of anidulafungin (AFG) against 32 mold isolates showed an excellent correlation between disk diffusion (DD) and broth microdilution methods. Based on our data, a 2-microg disk of AFG and a 24-h reading time might represent the best parameters for AFG DD testing against filamentous fungi.

Posaconazole against Candida glabrata isolates with various susceptibilities to fluconazole.

We investigated the in vitro activities of posaconazole (POS), fluconazole (FLC), amphotericin B (AMB), and caspofungin (CAS) against four clinical isolates of Candida glabrata with various susceptibilities to FLC (FLC MICs ranging from 1.0 to >64 microg/ml). POS MICs ranged from < or =0.03 to 0.5 microg/ml; AMB MICs ranged from 0.25 to 2.0 microg/ml, while CAS MICs ranged from 0.03 to 0.25 microg/ml. When FLC MICs increased, so did POS MICs, although we did not observe any isolate with a POS MIC greater than 0.5 mug/ml. Time-kill experiments showed that POS, FLC, and CAS were fungistatic against all isolates, while AMB at eight times the MIC was fungicidal against three out of four isolates of C. glabrata tested. Then, we investigated the activity of POS in an experimental model of disseminated candidiasis using three different isolates of C. glabrata: one susceptible to FLC (S; FLC MICs ranging from 1.0 to 4.0 microg/ml; POS MIC of < or =0.03 microg/ml), one susceptible in a dose-dependent manner (SDD; FLC MICs ranging from 32 to 64 microg/ml; POS MICs ranging from 0.125 to 0.25 microg/ml), and another one resistant to FLC (R; FLC MIC of >64 microg/ml; POS MIC of 0.5 microg/ml). FLC significantly reduced the kidney burden of mice infected with the S strain (P = 0.0070) but not of those infected with the S-DD and R strains. POS was significantly effective against all three isolates at reducing the kidney fungal burden with respect to the controls (P ranging from 0.0003 to 0.029). In conclusion, our data suggest that POS may be a useful option in the management of systemic infections caused by C. glabrata. Additionally, the new triazole may be a therapeutic option in those cases where an FLC-resistant isolate is found to retain a relatively low POS MIC.

Posaconazole activity against Candida glabrata after exposure to caspofungin or amphotericin B.

We evaluated the effects of sequential therapy with caspofungin (CAS) or amphotericin B (AMB) followed by posaconazole (POS) against Candida glabrata. The susceptibilities to POS of yeast cells pre-exposed to CAS or AMB were identical to those of untreated cells as shown by standard Clinical and Laboratory Standards Institute broth dilution, cell viability, and disk diffusion methods. We then investigated the activity of sequential regimens in an experimental model of disseminated candidiasis. CAS given at 1 mg/kg/day for 2 days followed by POS at either 15 or 30 mg/kg/day significantly reduced the counts compared to the controls, but this treatment was not superior to the use of CAS alone. Also, sequential regimens with AMB given at 1 mg/kg/day for 2 days followed by POS (AMB/POS) were effective at reducing the fungal burden against the controls. In addition, AMB/POS with both doses of the triazole were significantly more effective than AMB alone. Overall, our data showed that there is no therapeutic advantage in using CAS followed by POS, whereas an induction therapy with AMB followed by a maintenance regimen with POS might be a suitable strategy in managing C. glabrata infections.

Comparison between disk diffusion and microdilution methods for determining susceptibility of clinical fungal isolates to caspofungin.

We compared the caspofungin (CAS) susceptibility testing results generated by the disk diffusion (DD) assay with the results of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BD) reference method for 106 yeast isolates. The isolates represented 11 different fungal species, including Candida albicans (n = 50), C. parapsilosis (n = 10), C. glabrata (n = 10), C. tropicalis (n = 10), C. guillermondii (n = 6), C. rugosa (n = 5), C. krusei (n = 5), C. kefyr (n = 2), C. pelliculosa (n = 2), Saccharomyces cerevisiae (n = 3), and Geotrichum candidum (n = 3). The DD assay was performed in supplemented Mueller-Hinton agar with CAS, which was tested at concentrations of 2, 10, and 25 mug per disk. MICs and inhibition zone diameters were evaluated at 24 and 48 h. In general, the results obtained by the DD assay correlated well with those obtained by the BD method. In particular, a significant correlation between methods was observed when CAS was used at concentration of 2 mug/disk at a reading time of either 24 or 48 h.

Caspofungin in combination with amphotericin B against Candida parapsilosis.

Candida parapsilosis has emerged as an important nosocomial pathogen. In the present study, a checkerboard broth microdilution method was performed to investigate the in vitro activities of caspofungin (CAS) in combination with amphotericin B (AMB) against three clinical isolates of C. parapsilosis. Although there was a significant reduction of the MIC of one or both drugs used in combination, an indifferent interaction (fractional inhibitory concentration index greater than 0.50 and less than or equal to 4.0) was observed in 100% of cases. This finding was confirmed by killing curve studies. By a disk diffusion assay, the halo diameters produced by antifungal agents in combination were often significantly greater than those produced by each drug alone. Antagonism was never observed. In a murine model of systemic candidiasis, CAS at either 0.25 or 1 mg/kg/day combined with AMB at 1 mg/kg/day was significantly more effective than each single drug at reducing the colony counts in kidneys. Higher doses of the echinocandin (i.e., 5 and 10 mg/kg/day) combined with the polyene did not show any advantage over CAS alone. Overall, our study showed a positive interaction of CAS and AMB against C. parapsilosis.

Posaconazole prophylaxis in experimental systemic zygomycosis.

Three isolates of zygomycetes belonging to two different genera (Rhizopus oryzae and Absidia corymbifera) were used to produce a systemic infection in neutropenic mice. On days -2 and -1 and at 2 h prior to infection, the mice received either posaconazole (POS) at doses ranging from 20 to 80 mg/kg of body weight/day or amphotericin B (AMB) at 1 mg/kg/day. Antifungal drug efficacy was assessed by determination of the prolongation of survival, determination of the percentage of infected organs (brain, lung, spleen, and kidney), and histological examination for the number of infection foci and their sizes in brain and kidney tissues. AMB significantly prolonged the survival of mice infected with all isolates. POS significantly prolonged the survival of mice infected with zygomycetes. Cultured organs from mice infected with R. oryzae were all positive, while treated mice challenged with A. corymbifera generally showed lower percentages of infected organs compared with the percentages for the controls. Zygomycete isolates established an active infection (the presence of hyphae) in the brains and the kidneys of all controls. In mice challenged with R. oryzae, both antifungal drugs were effective at reducing the number and the size of infection foci in the kidneys. Only AMB reduced the numbers, but not the sizes, of infection foci in the brain. Finally, both drugs significantly reduced the numbers and the sizes of infection foci in both tissues of mice infected with A. corymbifera. Our data suggest that prophylaxis with POS has some potential to prevent zygomycosis.

Effects of caspofungin against Candida guilliermondii and Candida parapsilosis.

The in vitro activity of caspofungin (CAS) was investigated against 28 yeast isolates belonging to Candida albicans (n = 5), Candida guilliermondii (n = 10), and Candida parapsilosis (n = 13). CAS MICs obtained by broth dilution and Etest methods clearly showed a rank order of susceptibility to the echinocandin compound with C. albicans > C. parapsilosis > C. guilliermondii. Similarly, time-kill assays performed on selected isolates showed that CAS was fungistatic against C. albicans and C. parapsilosis, while it did not exert any activity against C. guilliermondii. In a murine model of systemic candidiasis, CAS given at doses as low as 1 mg/kg of body weight/day was effective at reducing the kidney burden of mice infected with either C. albicans or C. guilliermondii isolates. Depending on the isolate tested, mice infected with C. parapsilosis responded to CAS given at 1 and/or 5 mg/kg/day. However, the overall CFU reduction for C. guilliermondii and C. parapsilosis was approximately 100-fold less than that for C. albicans. Our study shows that CAS was active in experimental systemic candidiasis due to C. guilliermondii and C. parapsilosis, but this activity required relatively high drug dosages.

Candida guilliermondii fungemia in patients with hematologic malignancies.

The microbiological, clinical, and epidemiological features of most non-Candida albicans Candida species are well known, but much less is known about species such as Candida guilliermondii, an uncommon pathogen causing a variety of deep-seated infections in immunocompromised hosts. To characterize C. guilliermondii fungemia in patients with hematological malignancies and its susceptibility to antifungal drugs, all cases of C. guilliermondii fungemia diagnosed in our department between 1983 and 2005 were retrospectively analyzed and the literature was reviewed. C. guilliermondii caused 29/243 (11.7%) candidemia episodes diagnosed during the study period. Central venous catheters were the documented sources of candidemia in 19/29 episodes (65.5%), and invasive tissue infections were documented in 2 (6.9%). In the remaining eight, the catheter was not removed and the source of the fungemia remained obscure. Seven episodes ended in death, but only one could be attributed to invasive C. guilliermondii infection. Molecular typing data reveal no evidence of common infection sources. Isolates displayed high rates of in vitro susceptibility to amphotericin B (100%), voriconazole (95%), and fluconazole (90%) and lower rates of in vitro susceptibility to flucytosine (86%), itraconazole (76%), and caspofungin (33%). Our literature review confirms that C. guilliermondii is a significantly more frequent cause of candidemia among cancer patients compared with the general hospital population. It accounted for <1% of the total number of Candida bloodstream isolates reported in the articles we reviewed, with higher rates in Europe (1.4%) and Asia (1.8%) compared with North America (0.3%).

Comparison of the fungicidal activities of caspofungin and amphotericin B against Candida glabrata.

We investigated the fungicidal activity of caspofungin (CAS) and amphotericin B (AMB) against 16 clinical isolates of Candida glabrata. The minimum fungicidal concentrations (MFCs) of CAS were similar to those of AMB, ranging from 2.0 to >8.0 microg/ml. Time-kill assays performed on selected isolates showed that AMB was fungicidal at concentrations four times the MIC while CAS was not. A neutropenic-mouse model of disseminated infection was utilized to determine the residual fungal kidney burden. While doses as low as 0.3 and 1 mg/kg of body weight/day of CAS and AMB, respectively, were effective at reducing the counts with respect to controls, organ sterilization was reached when both drugs were administered at 5 mg/kg/day. Our study reveals that, similar to AMB, CAS has the potential for a fungicidal effect in vivo against this difficult-to-treat fungal pathogen.

Efficacy of caspofungin against Aspergillus terreus.

We investigated the in vitro and in vivo activities of caspofungin against Aspergillus terreus. The drug increased survival and reduced tissue fungal burden in neutropenic mice. Therefore, our data support the role of caspofungin in treating systemic infections due to this emerging pathogen.

Caspofungin in combination with amphotericin B against Candida glabrata.

The effects of caspofungin combined with amphotericin B were investigated with Candida glabrata. Although in vitro experiments showed an indifferent interaction, the combination regimen was the only therapeutic approach yielding organ sterilization in a murine candidemia model.

Tolerance to amphotericin B in clinical isolates of Candida tropicalis.

A broth microdilution method was used for testing amphotericin B against 33 clinical isolates of Candida tropicalis. All isolates were in vitro susceptible to the polyene (MIC [minimal inhibitory concentration] < or = 1.0 microg/mL). However, when the isolates were cultured in a medium containing amphotericin B at a concentration of 1.5 microg/mL, a wide interstrain variation of growth rate was observed. Five isolates (15%) proved to be highly tolerant to the drug and grew at a frequency ranging from 1 x 10(-1) to 2 x 10(-2). Twenty-three isolates (70%) grew at a frequency ranging from 1 x 10(-5) to 1 x 10(-8). The remaining five isolates (15%) failed to grow in drug-containing medium. In general, this growth variation was not associated with amphotericin B MICs displayed by the single isolates. In addition, the strains grown in drug-containing medium did not represent amphotericin B-resistant mutants, as shown by the maintenance of MICs similar to those of their respective parent isolates. Killing experiments conducted in selected isolates confirmed a variation of fungicidal activity of amphotericin B. To see whether this phenomenon was associated with a variation of amphotericin B response in vivo, we established an experimental model of systemic murine candidiasis in CD1 mice by intravenous injection of cells belonging to Candida tropicalis 3147 (growth rate at a frequency of 1 x 10(-1) in amphotericin B medium) and Candida tropicalis 4055 (no growth). Low (0.3 mg/kg/day) and high (1 mg/kg/day) doses of amphotericin B were both effective at reducing the fungal burdens in the kidneys of mice infected with either strain (p, 0.01 to 0.02). However, whereas the burden of mice infected with isolate 3147 and treated with the polyene at 0.3 mg/kg/day was reduced by 1.2 +/- 0.25 (mean +/- standard deviation) log10 cfu/g compared to untreated mice, the same dosing regimen yielded a burden reduction of 2.6 +/- 0.07 log10 cfu/g in mice infected with isolate 4055 (p < 0.001). Similarly, amphotericin B at 1 mg/kg/day yielded a burden reduction of 1.8 +/- 0.20 vs. 2.5 +/- 0.30 log10 cfu/g in mice infected with isolates 3147 and 4055, respectively (p < 0.001). Our data revealed a variable pattern of tolerance to amphotericin B among isolates of Candida tropicalis and showed that this phenomenon might influence the rate of organ clearance during therapy.

Sequential therapy with caspofungin and fluconazole for Candida albicans infection.

A sequential therapy of caspofungin (CAS) and fluconazole (FLC) administration for treatment of Candida albicans infection was investigated. Treatment with CAS followed by FLC was as effective as CAS treatment given alone for the same duration. Our data suggest that switching from CAS to FLC is a potentially explorable therapeutic option for treatment of systemic candidiasis.