JAC Advance Access originally published online on September 14, 2006
Journal of Antimicrobial Chemotherapy 2006 58(5):973-979; doi:10.1093/jac/dkl378
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Comparison of antifungal treatments for murine fusariosis
1 David Geffen School of Medicine at UCLA Los Angeles, CA, USA 2 Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center Torrance, CA, USA 3 Charles River Laboratories Davis, CA, USA 4 California State Polytechnic University Pomona, CA, USA
*Corresponding author. Tel: +1-310-222-6424; Fax: +1-310-782-2016; E-mail: ibrahim{at}labiomed.org
Received 30 May 2006; returned 2 August 2006; revised 3 August 2006; accepted 17 August 2006
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Abstract |
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Objectives: Fusarium solani infections are notoriously difficult to treat. We compared the efficacy of polyenes and an echinocandin in treating murine fusariosis to identify the optimal therapeutic regimen.
Methods: Neutropenic mice infected intravenously with F. solani were treated with amphotericin B (AmB), liposomal AmB (LAmB), amphotericin B lipid complex (ABLC), caspofungin acetate or a combination of LAmB and caspofungin. Treatment was initiated prior to infection (prophylactic therapy), 24 h post-infection (delayed therapy) or 2 days before infection and continued for 1 day after (continuous therapy).
Results: Prophylaxis only with LAmB significantly reduced brain or kidney fungal burden compared with placebo. No prophylactic treatment improved survival. LAmB levels in the kidneys were higher than ABLC or AmB levels, which were often undetectable. In the delayed therapy model, neither polyenes nor caspofungin improved survival. In the continuous therapy model, LAmB or LAmB plus caspofungin did not improve survival even though they did decrease fungal burden. In contrast, continuous caspofungin at 1 but not 5 mg/kg/day improved survival, but did not decrease fungal burden. Kidney inflammation and tissue necrosis were markedly decreased in mice treated with caspofungin compared with other treatments.
Conclusions: These studies demonstrate a dissociation between survival and tissue fungal burden during murine fusariosis. Although prophylactic LAmB may be useful at reducing tissue fungal burden, polyenes had limited survival benefit for active fusariosis. Caspofungin at 1 but not 5 mg/kg/day mediated surprising improvements in survival during active fusariosis, despite lack of reduction in fungal burden. Further studies are warranted.
Keywords: mice , antifungal therapy , Fusarium solani
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Introduction |
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Fusariosis is an increasingly common cause of life-threatening infections in highly immunocompromised patients, particularly in the setting of haematopoietic stem cell transplantation.1 The infection is caused by members of 20 species among the genus Fusarium, of which the most common human pathogens are Fusarium solani complex.1,2 Indeed, F. solani complex accounts for approximately half of the reported infections, followed by Fusarium oxysporum, Fusarium moniliforme and Fusarium verticillioides, each of which account for 10–14% of the total incidence of infections.3–7 Based on data from animal models,8 the higher incidence of F. solani infection might reflect an increased virulence of this species. Fusariosis can be manifested as localized or disseminated infection, with the status of the host and the degree of tissue invasion the most critical predictors of outcome.1 To date, clinical trials of fusariosis have been problematic due to the relative rarity of the disease. As a result, optimal antifungal therapy for fusariosis remains unclear, and preclinical data have been used as an indicator of potentially useful therapeutic approaches.
In vitro, F. solani is among the most resistant fungi to current antifungal drugs. Indeed, the activity of antifungal agents, including polyenes, against F. solani is disappointing.9 Because in vitro activity might not always correlate with in vivo efficacy, we compared the in vivo efficacy of different antifungal agents to determine the optimal therapeutic strategy for prophylactic, continuous and delayed therapy of F. solani infection in mice.
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Materials and methods |
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Organisms and culture conditions
F. solani 95–2478 is a blood isolate provided by Dr P. Ferrieri (University of Minnesota). The organism was grown on potato dextrose agar (PDA) for 5 days at room temperature. Macroconidia were collected in endotoxin-free PBS containing 0.01% Tween 80, washed with PBS, counted with a haemocytometer and adjusted to the desired concentration in endotoxin-free PBS.10
Drugs and therapy regimens
Amphotericin B (AmB), liposomal AmB (LAmB) and amphotericin B lipid complex (ABLC) were diluted in 5% dextrose water. Pure powder of caspofungin was dissolved in sterile distilled water. In all models, mice were infected via the tail vein with F. solani. In all studies control groups were treated with placebo, using the diluent, 5% dextrose in water.
Animal model
BALB/c male mice (
20 g, National Cancer Institute) were rendered neutropenic by cyclophosphamide. For experiments in which mice were sacrificed prior to day +3 relative to infection, a single dose of cyclophosphamide (200 mg/kg) intraperitoneally (ip) was administered on day –2 relative to infection. For experiments in which mice were kept past day +3 relative to infection, two doses of cyclophosphamide were administered, at 200 mg/kg on day –2 relative to infection and at 150 mg/kg on day +3 relative to infection. In a pilot study, total leucocyte counts were determined with the Unopette system per the manufacturer's instructions (Fisher Scientific, Hampton, N.H.), confirming that mice were rendered pancytopenic for approximately 11 days by this cyclophosphamide regimen (Figure 1).
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Mice were infected through the tail vein with the appropriate inocula of F. solani conidia in 0.2 mL endotoxin-free PBS 2 days after the first dose of cyclophosphamide. To confirm the inocula, dilutions were streaked on PDA plates and colonies were counted following a 48 h incubation period at room temperature. The primary efficacy endpoint was time to death. As a secondary endpoint, kidney and brain fungal burdens were determined by homogenization by rolling a pipette on organs placed in Whirl-Pak bags (Nasco, Fort Atkinson, WI, USA) containing 2 mL saline. Kidneys and brains were harvested because pilot studies confirmed that these were the primary and secondary target organs, respectively. The homogenate was serially diluted in 0.85% saline and then quantitatively cultured on PDA. Values were expressed as log10 cfu/g tissue.
Kidney histopathology was also performed. Briefly, kidneys were collected 72 h post-infection, fixed in 10% zinc-buffered formalin, paraffin-embedded, sectioned and stained with haematoxylin and eosin (H&E). Sections (5 µm thick) were examined by a Board certified veterinary pathologist. All procedures involving mice were approved by the institutional animal use and care committee, following the National Institutes of Health guidelines for animal housing and care.
Paecilomyces variotti agar inhibition assay for tissue drug levels
To determine tissue drug levels, a bioassay was utilized. Paecilomyces variotti (ATCC #22319) was cultured on PDA media (Difco Labs) at 40°C for 48 h in the dark to stimulate spore production, followed by incubation for 72 h at room temperature.11 Spores harvested in saline containing 0.01% Tween 80 were filtered through sterile gauze and the suspension sonicated for 1 min in a water bath sonicator to break up clumps. Counts were determined with a haemocytometer. The spore suspension was added to sterile molten Antibiotic Media 19 agar (AM19a, Difco Labs) to get a final concentration of 3 x 104 spores/mL and poured into Petri dishes. After solidification of seeded media, agar wells were made using a 7-well circular template. Standard solutions of amphotericin B (Spectrum Chemical Manufacturing Corp) in DMSO (Sigma Chemical) diluted in PBS, and ABLC and LAmB diluted 1:1 with methanol, heated for 10 min at 65°C, and diluted further in PBS, were prepared. Weighed tissue samples were homogenized (Tissue Tearor, 7mm microtip probe) in 1 mL of PBS, diluted 1:1 with methanol and heated for 10 min at 65°C. Samples were then centrifuged at 950 g for 8 min and the supernatant diluted in PBS. Each test sample dilution or drug standard dilution (150 µL) was placed in an agar well, incubated for 1 h at room temperature and then incubated at 35°C for 20 h. Tests were done in duplicate. The zone of inhibition around each well was measured with a Vernier caliper and average zone size used to determine drug concentration in the tissue samples (µg/g) using linear regression analysis.
Statistical analysis
The non-parametric log rank test was used to determine differences in survival times of the mice. Differences in tissue fungal burdens and tissue drug levels in the infected organs were compared by the Mann–Whitney U-test, or non-parametric Steel test for multiple comparisons. P values of <0.05 were considered significant.
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The kidneys are rapidly infected during murine disseminated fusariosis, while brain infection occurs more gradually
As a foundation for determining the impact of antifungal therapy on F. solani fungal burden, we began by determining the kinetics of kidney and brain infection in the neutropenic mouse model [single dose 200 mg/kg of cyclophosphamide ip 2 days before infection]. Neutropenic mice were infected with 3 x 105 spores of F. solani and sacrificed at 5, 24 and 48 h post-infection. Heavy kidney infection was detected at 5 h post-infection, and the kidney fungal burden increased during the first 48 h (Figure 2). In contrast, brain fungal burden was not detectable in most mice by 5 and 24 h post-infection. By 48 h post-infection, the majority of mice had developed detectable brain fungal burden.
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Only prophylactic LAmB reduced tissue fungal burden, and no therapy improved survival
To determine if prophylactic antifungal therapy could reduce tissue fungal burden, mice were treated intravenously (iv) with placebo or 60 mg/kg of LAmB or ABLC over 4 weeks (i.e. 5 mg/kg three times per week x 4 weeks). Prophylactic AmB was administered ip five times per week for 4 weeks at 3 mg/kg ip per dose to avoid sclerosing the tail vein, which would occur with multiple weeks of iv AmB treatment. One day following the last dose of antifungal drug, mice were treated with cyclophosphamide and 2 days later they were infected with F. solani (i.e. 3 days after the last dose of antifungal drug). Organs were collected 48 h post-infection. Mice receiving LAmB had significant reductions in their kidney and brain fungal burden compared with control (Figure 3a). No other group had significant reductions in tissue fungal burden compared with control.
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P = 0.07 versus control. Bars = medians.The experiment was then repeated with infection of the mice 7 days following the last dose of the antifungal drug (again 2 days after the single cyclophosphamide dose). Therefore, cyclophosphamide was administered 5 days after the last dose of antifungal drug, with mice infected 2 days later. Again only LAmB decreased brain fungal burden, and there was a trend to reduced kidney fungal burden (P = 0.07) (Figure 3b).
To provide additional context for prophylactic antifungal efficacy, we determined the tissue concentrations of antifungal agents in brains and kidneys of infected mice. As above, a single cyclophosphamide dose was administered on day –2 relative to infection. Infection was carried out either 3 or 7 days after the last antifungal treatment and organs were collected 48 h post-infection (i.e. 5 or 9 days after the last antifungal treatment). LAmB was detectable in kidneys at both 5 or 9 days after the last dose of antifungal therapy, whereas ABLC was not detected in kidneys at either time point (Table 1). AmB was detected in the kidneys at 5 but not 9 days after the last dose of antifungal therapy. None of the drugs was detectable in brains at either time point (Table 1).
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To determine if the reductions in tissue fungal burden translated into improved outcome, we determined the effect of prophylactic antifungal therapy on survival of neutropenic mice with disseminated fusariosis. Mice were treated with LAmB, ABLC, AmB or placebo over 4 weeks, as above. Mice were infected via the tail vein with F. solani 3 days after the last dose of antifungal drug. In this experiment, two doses of cyclophosphamide were administered, on day –2 (200 mg/kg) and day +3 (150 mg/kg) relative to infection (which was 1 day and 6 days after the last dose of antifungal drug). None of the treatments improved survival compared with placebo (Figure 4).
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When administered after established infection, no antifungal therapy improved survival of neutropenic mice with disseminated fusariosis
To determine the relative efficacies of antifungal therapies for established fusariosis infection, mice were treated with two doses of cyclophosphamide as above and infected 2 days after the first cyclophosphamide dose with 106 spores of F. solani. The infected mice were treated iv with AmB (1 mg/kg/day), LAmB (5 or 10 mg/kg/day), ABLC (5 or 10 mg/kg/day) or caspofungin (1, 5, 10 mg/kg/day) from days 1 through 5 post-infection. No antifungal therapy improved survival compared with placebo (data not shown).
When administered as continuous therapy, only caspofungin at 1 mg/kg/day improved survival of neutropenic mice with disseminated fusariosis
We have previously found that combination antifungal therapy with caspofungin and a polyene was synergistic in a diabetic, ketoacidotic murine model of disseminated Rhizopus oryzae infection.12 We therefore compared the efficacy of antifungal therapy with LAmB (15 mg/kg/day), caspofungin (1 or 5 mg/kg/day) or LAmB (15 mg/kg/day) + caspofungin (1 mg/kg/day) initiated 2 days prior to infection, given on the day of infection, and continued for 1 day after infection (i.e. continuous therapy = 4 daily doses) in mice treated with two doses of cyclophosphamide as above. Only caspofungin monotherapy at 1 mg/kg/day improved survival (Figure 5).
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To explore the mechanism of protection in this model, we determined tissue fungal burden and evaluated tissue histopathology. Tissue fungal burden was evaluated on-therapy (24 post-infection, after 4 doses of antifungal therapy) and after the end of antifungal therapy (72 h post-infection, 48 h after the last dose of antifungal therapy). In contrast to the survival results, at 24 h post-infection continuous therapy with LAmB or LAmB plus caspofungin reduced both brain and kidney fungal burden, whereas caspofungin at 1 mg/kg/day had no effect on tissue fungal burden (Figure 6). However, regrowth of Fusarium off therapy was rapid; by 72 h post-infection, which was 48 h after the end of antifungal therapy, fungal burden was no longer less than in placebo-treated animals.
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By histopathology, treatment with LAmB, combination LAmB plus caspofungin or placebo resulted in marked inflammatory infiltrate in kidneys with resultant necrosis (Figure 7). In contrast, mice treated with caspofungin monotherapy had minimal inflammation and tissue degeneration or necrosis.
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Discussion |
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Clinical F. solani infections are notoriously difficult to treat.1 Similarly, we found that no antifungal therapy improved the survival of mice when administered prophylactically, or for established disseminated fusariosis. It was surprising that continuous therapy with caspofungin resulted in improved survival, even though Fusarium species are resistant to echinocandins by standard susceptibility testing in vitro.13–16 These data are akin to the efficacy of caspofungin for murine mucormycosis, another mould which is resistant to echinocandins by standard susceptibility testing.12,17
Despite the fact that neither prophylactic nor continuous LAmB improved survival, both prophylactic and continuous LAmB reduced tissue fungal burden. Furthermore, despite the fact that continuous caspofungin therapy did improve survival, it did not reduce tissue fungal burden. Finally, the combination of continuous caspofungin + LAmB therapy was indifferent with respect to tissue fungal burden (showing reduced tissue fungal burden comparable to LAmB), but was antagonistic with respect to survival (showing no improvement in survival, again comparable to LAmB). Therefore, our results clearly indicate a dissociation between the effect of antifungal therapy on tissue fungal burden and survival in the murine model of fusariosis, as therapies that reduced tissue fungal burden did not improve survival and therapies that improved survival did not significantly reduce tissue fungal burden. These data suggest that survival outcome depended on other factors, such as drug toxicity, alterations in host inflammatory response or possibly alterations in mycotoxin production by the fungus. Indeed, tissue histopathology results clearly indicated greater inflammatory responses and necrosis in the kidneys of infected mice treated with LAmB, LAmB + caspofungin or placebo as compared with caspofungin.
Fusarium species are known for their ability to produce a variety of mycotoxins, of which the major class is called fumanisins.18 These mycotoxins are suspected to contribute to the pathogenesis of fusariosis.1 In this respect, and aside from its potential interactions with the host inflammatory response, it is possible that caspofungin alters virulence of F. solani, possibly by modulating production of mycotoxins. We have previously found that caspofungin mediated surprising survival benefit during murine zygomycosis, despite the fact that generally it did not significantly reduce tissue fungal burden.17,19 The role of caspofungin in altering mycotoxin production by R. oryzae or F. solani is under active investigation.
It is not surprising that neutrophils are detected in tissues despite confirmed peripheral blood neutropenia. The kinetics of neutrophil survival differs in blood and at the site of infection in tissues, and we have previously reported the ready detection of inflammatory neutrophil infiltrates in the tissues of mice with concurrent peripheral blood leucopenia due to cyclophosphamide therapy.20
We and others have previously found that the in vivo activity of caspofungin against Aspergillus21 and R. oryzae17 demonstrates an inverse dose–response relationship, with maximal activity at 1 mg/kg/day, and enhancement of infection at 10 mg/kg/day. Similarly, Stevens et al.22 found that higher concentrations of caspofungin are less active in vitro against Candida albicans. Finally, micafungin, a related echinocandin, has been shown to have a similar diminished efficacy against fungal infections at higher concentrations.23 Our results with caspofungin against F. solani are concordant, with improvement in survival with caspofungin continuous therapy only evident at the 1 mg/kg/day dose, and not at the 5 mg/kg/day dose.
Very few studies have compared the activity of LAmB versus ABLC head to head. We found that LAmB mediated a greater reduction in tissue fungal burden in vivo. However, neither LAmB nor ABLC mediated improved survival when administered prophylactically or for established fusariosis.
In summary, we found that reductions in tissue fungal burden did not translate into improved survival during murine fusariosis. Caspofungin mediated a surprising survival benefit during continuous therapy, but only when dosed at 1 mg/kg/day. Our results suggest that other factors contribute to survival during disseminated fusariosis, such as the severity of tissue inflammation and possibly mycotoxin production. Further investigations to better delineate the cause of death during fusariosis and the mechanisms of benefit of caspofungin therapy are warranted.
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None to declare.
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Acknowledgements |
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This work was presented in part at the Forty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2005 (Abstract # B-49). This study was supported by a research and educational grant from Gilead Sciences Inc. Research described in this manuscript was conducted in part at the research facilities of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center.
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References |
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