Efficacy of Albaconazole (UR-9825) in Treatment of Disseminated Scedosporium prolificans Infection in Rabbits

There are no effective therapeutics for treating invasive Scedosporiumprolificans infections. Doses of 15, 25, and 50 mg/kg of bodyweight/day for the new triazole albaconazole (ABC) were evaluatedin an immunocompetent rabbit model of systemic infection withthis mold. Treatments were begun 1 day after challenge and givenfor 10 days. ABC at any dose was more effective than amphotericinB (AMB) at 0.8 mg/kg/day at clearing S. prolificans from tissue(P < 0.007). The percentages of survival at 25 mg of ABC/kg/daywere similar to those obtained with AMB. Rabbits showed 100%survival when they were treated with 50 mg of ABC per kg (P< 0.0001 versus control group), and only this dosage wasable to reduce tissue burden significantly in the five organsstudied, i.e., spleen, kidneys, liver, lungs, and brain.

INTRODUCTION

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Introduction

Scedosporium prolificans is an emerging filamentous fungus thatinfects both immunosuppressed and healthy patients, and it isthe most common agent of disseminated phaeohyphomycosis (2,11, 14, 15, 20). The infection is probably mostly acquired byinhalation of conidia and can take a wide variety of forms.Disseminated infection is associated mainly with patients withunderlying blood malignancies and is usually fatal. The infectionis generally treated with amphotericin B (AMB) and occasionallywith other drugs, although the outcome is not often successful(2, 9, 15, 20). Several in vitro studies have demonstrated thatS. prolificans is generally resistant to the antifungal agentsavailable, and this correlates with the poor clinical response(5, 7). In a previous paper, we demonstrated that high dosesof liposomal amphotericin B (10 mg/kg of body weight/day) combinedwith granulocyte colony-stimulating factor were moderately effectivein the treatment of murine scedosporiosis (18). However, furtherstudies are required to determine whether more appropriate dosagesof the drug and more suitable strategies that combine it withgranulocyte colony-stimulating factor or other lymphokines providebetter results. Another alternative, which is practically unexplored,is to use a therapy combining two antifungal agents. In vitrostudies have demonstrated that itraconazole combined with terbinafinehas a synergic effect against clinical isolates of this fungus(16). This is a line of investigation that deserves to be developed,although the rapid clearance of both drugs in mice makes itnecessary to use other animal models for testing this combination.In spite of these promising approaches, alternative treatmentsfor these severe and increasingly frequent mycoses still needto be investigated. The new triazole albaconazole (ABC) showedgood in vitro activity against this fungus in a recent studywhich tested 30 strains (5). This drug has also shown good invitro activity against dermatophytes, Aspergillus, Paecilomyces,and Candida species, among others (4, 8, 19). The concentrationsof this drug achieved in plasma of mice are very low, and itshalf-life is short; however, its pharmacokinetics and bioavailabilityare excellent in rabbits (1). The aim of the present study wasto develop a rabbit model of systemic infection by S. prolificansto evaluate the efficacy of an escalation in dose of ABC.

MATERIALS AND METHODS

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Materials and Methods

Strains. Two strains of S. prolificans, FMR 6654 and FMR 3569, isolatedfrom human blood, were used.

Animals. New Zealand White male rabbits (weight, ca. 2.5 kg) obtainedfrom San Bernardo S.L., Navarra, Spain, were used in the study.The rabbits were individually housed and were maintained withwater and standard rabbit feed ad libitum. They were monitoreddaily for 18 days. Conditions were approved by the Animal WelfareCommittee of the Rovira i Virgili University.

Inoculum preparation. The strains were retrieved from storage in slant cultures withsterile paraffin oil and were subcultured on potato dextroseagar plates at 30°C for 7 to 10 days. The inoculum was preparedas described in previous studies (18). Rabbits were infectedintravenously via a lateral ear vein.

Model infection. To determine a suitable lethal dose, groups of five rabbitswere established. Three inocula of two different strains weretested, i.e., 5 x 10⁶, 1 x 10⁷, and 1 x 10⁸ CFU of the strainFMR 6654/animal and 5 x 10⁶, 1 x 10⁷, and 5 x 10⁸ CFU of thestrain FMR 3569/animal. Animals were checked daily for 18 days.

Antifungal agents. ABC (Uriach & cía., Barcelona, Spain) was dissolvedin 0.2% carboxymethylcellulose and 1% Tween 80 in distilledwater at a concentration of 125 mg/ml. AMB (Fungizona; SquibbIndustria Farmaceutica, Barcelona, Spain) was dissolved in distilledwater for a final stock solution of 5 mg/ml. The final concentrationfor administration was prepared in 5% dextrose.

Treatments. After screening two strains of S. prolificans at different concentrationsof inocula, animals, in groups of eight, were infected withan inoculum of 10⁷ CFU of the strain FMR 6654. Treatments began24 h after infection and were given once daily for 10 days.ABC was administered orally with a 5-ml syringe with an attachedanimal feeding needle at 15, 25, and 50 mg/kg. AMB was administeredintravenously as a bolus, via a lateral ear vein, at 0.8 mg/kg.The control group received oral placebo treatment, i.e., 0.2%carboxymethylcellulose and 1% TWEEN 80 in distilled water.

Tissue burden study. Animals which experienced paralysis, convulsion, or prolongedanorexia or dehydration or survived to the end of the experimentwere sacrificed by injecting a concentrated pentobarbital solutionintravenously (22). Kidneys, spleen, liver, lungs, and brainwere collected, and portions of 1 g of each organ were homogenizedin 2 ml of 0.9% saline. Homogenates were serially diluted insaline and plated in duplicate on potato dextrose agar, andcolonies were counted after 96 h of incubation at 30°C.

Histopathology. A portion of each specimen was placed in 10% buffered formalinfor the histopathological study. Organs were sectioned in aparaffin block, and the sections were stained with hematoxylin-eosin,periodic acid Schiff, and Grocott methenamine silver (6).

Statistical methods. Survival rates were evaluated with the Kaplan-Meyer test usingGraph Pad Prism software, version 3.00 for Windows. Organ burdenswere converted to log₁₀ CFU and compared using the Mann-WhitneyU test.

RESULTS AND DISCUSSION

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Results and Discussion

In general mortality correlated with the size of the inocula.The largest inoculum (10⁸ CFU) of the strain FMR 6654 was highlylethal (100% mortality in 5 days) for rabbits with a mean survivaltime (MST) of 4 days. The rabbits infected with 10⁷ CFU diedbetween 4 and 10 days postinfection (MST = 7.13 days). Withthe smallest inoculum (5 x 10⁶ CFU), only two animals died in18 days. Rabbits infected with 5 x 10⁸ CFU of strain FMR 3569showed an MST of 2 days. Animals which received an inoculumof 10⁷ CFU of that strain had an MST of 11.5 days, and thoseinfected with 5 x 10⁶ CFU had 100% survival (MST > 18 days).

Inocula of 10⁷ for both strains were tested again in triplicate.The results obtained with the strain FMR 6654 were more reproducible(MST = 7.19 ± 0.32 days) than with strain FMR 3569 (MST= 9.58 ± 4.69 days). On the basis of these results, aninoculum of 10⁷ CFU of strain FMR 6654/animal was chosen forthe study.

Figure 1 shows the survival curves for the treated animals andthe control group. Untreated animals began to die 4 days postinfection,and by day 12 no animal survived (MST = 7.12). The mortalityof the animals treated with ABC at 15 mg/kg/day was similar(MST = 7.25) to that of the control group (P = 0.34). Animalstreated with ABC at 25 mg/kg/day or AMB at 0.8 mg/kg/day showed50% survival (MST = 13.37 and 14.87, respectively), and survivalrates were significantly greater than those for untreated animals(P < 0.01). All the animals that received ABC at 50 mg/kg/daysurvived at the end of the experiment (P < 0.0001 versuscontrol group results).

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FIG. 1. Survival curves of rabbits infected with 10⁷ CFU of S. prolificans FMR 6654.

CFU counts from semiquantitative cultures of spleen, kidney,liver, lung and brain are shown in Table 1. The organs of allthe untreated animals, in particular the kidneys, were extensivelyinfected. The animals treated with ABC at all tested doses hadlower tissue burdens than the control group. S. prolificanswas undetectable in the liver, spleen, and lung cultures. AMBreduced tissue burden significantly in the liver and spleen(P = 0.002 and 0.012 versus control group results, respectively),but ABC was more effective at all doses in the spleen (P <0.001 versus AMB group results). In lung tissue, AMB reducedcolony counts but not significantly (P = 0.061). However, allthe animals that received ABC showed negative cultures fromthis organ (P < 0.007 versus AMB results; P < 0.001 versusresults for untreated animals). In the kidney and brain, onlyanimals receiving ABC at a dose of 50 mg/kg/day significantlyreduced tissue burden in comparison to the control group (P< 0.001 and P = 0.023, respectively).

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TABLE 1. Semiquantitative results of organ cultures of rabbits treated with antifungal therapy begun 24 h after challenge

The histopathological study confirmed that the kidney was theorgan that was most affected, but no visible lesions were observedin the liver, lung, or spleen from either treated or untreatedanimals. In control animals, the kidney showed numerous micro-and macroabscesses with central necrotic areas, considerableinflammatory cell response, and numerous fungal elements inparenchymatous tissue (Fig. 2b) and the pelvis. Small areasof glomerular and tubular necrosis with hyphae and conidia wereobserved. Approximately 20% of control animals showed microabscessesin the brain (Fig. 2a). The histopathological features of theanimals treated with the lowest doses of ABC were similar tothose of the untreated group. No lesions, however, were foundin the brain. AMB or ABC at 25 mg/kg/day attenuated the histopathologicalseverity in the kidney the same as in controls. In both groups,abscesses with some inflammatory and fungal cells were observed.No histological damage was observed in the central nervous system.

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FIG. 2. (a) Subcortical microabscess in the brain of an untreated animal. Periodic acid Schiff stain (magnification, x60). (b) Fungal growth in parenchymatic tissue of kidney of an untreated animal. Grocott stain was used (magnification, x240). (c) Area of tissue regeneration after a necrotic process in renal parenchyma. Animal was treated for 10 days with ABC at 50 mg/kg/day. Periodic acid Schiff stain was used (magnification, x200).

The least renal affection was observed in animals that receivedABC at a rate of 50 mg/kg/day. In this group, only small residualfoci with few or no fungal cells were observed. It is worthmentioning that in the animals of this group, areas with postnecrotictissular regeneration, without inflammatory or fungal cells,were also present (Fig. 2c). No signs of toxicity were detectedin any of the treated animals.

Up to now murine models were the only models developed to studyScedosporium infections in animals (3, 17, 18). The mouse isusually the animal of choice for infections by numerous fungalspecies because of its availability and the large accumulationof information regarding its pathophysiology. However, in ourcase the pharmacokinetics of ABC in mice was not useful. Therapid clearance of ABC in mice makes these animals unsuitablefor testing efficacy. Consequently, we established a reproduciblemodel of acute scedosporiosis in rabbits and demonstrated thatthe severity of the infection is determined by the size of theinoculum. Although not as commonly used as mice, rabbits havebeen useful for testing several drugs against systemic infectionsby different species of filamentous fungi (12, 13, 21, 22).In the development of our rabbit model, we have followed someof the procedures used in such studies. In our model, the infectionwas especially acute in the kidneys and brain, where abscessesand hyphae were clearly evident. Previously, we had also noticedtropism of S. prolificans for these organs in mice (3). Theresults of our present study demonstrated that ABC has therapeuticefficacy against systemic scedosporiosis in nonimmunosuppressedanimals. This is important because the fungus causes infectionsnot only in neutropenic patients but also in immunocompetentpeople (10, 23). In the latter, the fungus usually causes boneand joint infections and locally invasive diseases. Infectionsby S. prolificans have increased dramatically in recent years,and it is now one of the most common filamentous fungi thatcause systemic infections in humans. In a recent review of disseminatedphaeohyphomycosis reported in the last decade, S. prolificanswas by far the most common species (20). The fact that evenlow doses of ABC were able to reduce tissue burden more effectivelythan AMB and that at higher doses rabbits reached 100% survivalis very hopeful, because so far there have been no effectivetreatments of systemic scedosporiosis.

FOOTNOTES

* Corresponding author. Mailing address: Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, 21 Sant Llorenç Street, 43201 Reus, Spain. Phone: 977 759 359. Fax: 977 759 322. E-mail: umb{at}fmcs.urv.es.

REFERENCES

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