1 Unitat de Microbiologia and 2 Unitat d'Anatomia Patòlogica, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spain
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Abstract |
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Methods: Mice were rendered neutropenic by intraperitoneal cyclophosphamide and intravenous (iv) 5-fluorouracil administration. Animals were infected with iv or intracranial inoculation of 1x104, 5x104 or 5x105 cfu of a clinical strain of S. apiospermum. Tissue burden reduction was determined in kidneys and brain 4 days after the infection. Efficacy of amphotericin B and liposomal amphotericin B (0.8 mg/kg/day intraperitoneally and 40 mg/kg/day iv, respectively) was evaluated in neutropenic mice infected iv or intracranially with 5x104 cfu. Survival was analysed with the log-rank test. Fungal burden values of different groups were compared using the MannWhitney U-test.
Results: In our model, intracranial infection produced a higher fungal load in the brain and a lower fungal load in the kidney than iv inoculation. Survival of animals infected intracranially and treated with amphotericin B or liposomal amphotericin B (mean survival time = 8.3 and 9.2 days, respectively) was not different from the control group (P=0.58 and 0.85, respectively).
Conclusions: We have developed a murine model of cerebral scedosporiosis, which may be useful for studying various pathological aspects of this infection and evaluating new therapeutic approaches. Amphotericin B and liposomal amphotericin B were unable to resolve the infection.
Keywords: Scedosporium apiospermum , brain infections , animal models
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Introduction |
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Materials and methods |
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A complementary survival study was performed to determine the efficacy of amphotericin B and liposomal amphotericin B on both iv and intracranial animal models. Amphotericin B deoxycholate (Fungizona; Squibb Industria Farmacéutica, Barcelona, Spain) and liposomal amphotericin B (AmBisome; Gilead Sciences S.A., Barcelona, Spain) were reconstituted with sterile distilled water and further diluted in 5% sterile dextrose solution to reach the desired concentrations for administration. Six groups of 10 immunodepressed mice per group were used to assay the efficacy of amphotericin B and liposomal amphotericin B. Infection was established iv or intracranially with 5x104 cfu/animal. Treatments were begun 1 day after infection and were administered daily for 10 days. Amphotericin B and liposomal amphotericin B were administered iv at 0.8 and 40 mg/kg/day, respectively. Two control groups, infected iv and intracranially, received glucose 5% iv as placebo for 10 days. Survival was analysed with the log-rank test and fungus burden values were converted into log10 cfu. Data were compared using the MannWhitney U-test. GraphPad Prism software for windows was used for statistical analysis.
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Results |
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The fungus proved to be highly lethal when it was inoculated iv. Figure 1(a) shows the survival percentages of infected mice. The inoculum of 1x104 cfu caused a mortality of 100% by day 11, with a mean survival time (MST) of 8 days. MST was shorter in animals infected with higher inocula, i.e. 5.8 days with an inoculum of 5x104 cfu and 3.6 days with an inoculum of 5x105 cfu. Tissue burden studies demonstrated a similar doseresponse, the kidneys being more affected than the brain (Figure 2). The mean cfu/g in the kidneys of the infected animals, from the lowest to the highest inoculum, were 2.15 ± 0.51, 2.57 ± 0.39 and 3.96 ± 0.43 log10 cfu/g, and 0.90 ± 0.54, 1.90 ± 0.32 and 2.17 ± 0.39 log10 cfu/g in the brain. A histopathological study showed low tissue infection in brain and kidneys with few microabscesses and/or inflammatory cells.
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The groups of mice infected intracranially were tested in duplicate. Since there were no statistical differences between the two tests for each inoculum, the results were grouped in a single curve (Figure 1b). Animals infected with 1x104 cfu showed symptoms of neurological infection, such as disorientation, convulsion and anorexia. However, they all survived to the end of the experiment (day 14 post-infection). By contrast, higher inocula, such as 5x104 and 5x105 cfu/animal, caused 100% mortality (MST of 8.5 and 3.9 days, respectively). Tissue burden correlated with the results obtained in the survival study. Fungal load was significantly higher in the brain with the lowest inoculum than in the kidneys with the highest inoculum (Figure 2). Animals infected with the lowest inoculum showed 2.57 and 1.03 log10 cfu/g in the brain and kidneys, respectively. cfu counts were higher with higher inocula, i.e. 3.38 and 1.10 log10 cfu/g in the brain and kidneys, respectively, when the inoculum was 5 x 104 cfu/g, and 3.97 and 1.56 log10 cfu/g, respectively, when it was 5 x 105 cfu/animal. The histopathological study revealed the presence in the brain of macro- and microabscesses with necrosis and hyphae (conidia were not observed) (Figure 3). The number and severity of the lesions increased with the size of the inoculum.
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The MST of control animals infected iv or intracranially were 5.8 and 8.5 days, respectively. In the iv model, treatments with amphotericin B or liposomal amphotericin B (Figure 4) increased the MST (6.4 and 7.2 days, respectively) but not significantly in comparison with the control group (P=0.43 and P=0.072, respectively). Animals infected intracranially and treated with amphotericin B or liposomal amphotericin B did not show increased survival (MST = 8.3 and 9.2 days, respectively) in comparison with the control group (P=0.58 and 0.85, respectively).
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Discussion |
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Acknowledgements |
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Footnotes |
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References |
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