a Unitat de Microbiologia, Facultat de Medicina and Institut d'Estudis Avançats; b Laboratori de Microbiologia, Hospital Universitari de Sant Joan de Reus, Reus; c Servei de Patologia, Hospital Universitari de Tarragona Joan XXIII, Tarragona; d Unitat de Medicina Preventiva, Facultat de Medicina, Universitat Rovira i Virgili, Tarragona, Spain
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Abstract |
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Introduction |
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Materials and methods |
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S. prolificans FMR 3569 isolated from a patient with disseminated infection in Spain, was cultured on potato dextrose agar (PDA) plates for 710 days at 30°C. The inocula were prepared by flooding the surface of the agar plate with saline solution, scraping the sporulating mycelium with a culture loop and drawing up the resultant suspension with a sterile Pasteur pipette. The suspensions were then filtered once through a sterile gauze to remove hyphae. The numbers of conidia in the suspensions were counted with a haemocytometer and verified by plating dilutions of the suspensions on Sabouraud dextrose agar (SDA).
Animals
OF1 male mice (Charles River, Criffa S.A., Barcelona, Spain) with a mean weight of 30 g were used. Animals were housed in standard boxes with corncob bedding and free access to food and water. All animal care procedures were supervised and approved by the Universitat Rovira i Virgili Animal Welfare Committee.
Drugs
AMB (purchased as Fungizone; Squib Industria Farmacéutica, S.A., Barcelona, Spain), LAMB (provided as AmBisome by NeXstar Farmacéutica S.A., Madrid, Spain) and recombinant human G-CSF (purchased as Granulokine; Laboratorios Pensa, Barcelona, Spain) were used. The three compounds were diluted in 5% dextrose solution to reach the desired concentrations.
Immunosuppression
Mice were immunosuppressed with a single dose of cyclophosphamide 200 mg/kg ip (purchased as Genoxal; Laboratorios Funk S.A., Barcelona, Spain) and with 5-fluorouracil 150 mg/kg iv (purchased as Fluoro-uracil; Productos Roche S.A., Madrid, Spain) on the day of infection.
Infection
In a series of pilot experiments, 0.2 mL of four conidial suspensions (2.5 x 104, 2.5 x 105, 2.5 x 106 and 2.5 x 107 conidia/mL) were injected via a lateral tail vein of mice to determine (i) the inoculum that provoked a lethal infection that resulted in 100% mortality by days 1012 for the survival study, and (ii) the inoculum that provoked a weaker infection, with a 50% survival by day 10, for the tissue burden studies.
Treatment
For the survival study 150 mice were infected with 0.2 mL of a suspension of S. prolificans 2.5 x 105 conidia/mL. The inoculated mice were randomly assigned to five treatment groups of 30 animals. Each group received a different treatment, i.e., AMB, LAMB, G-CSF and LAMB + G-CSF, the fifth being the untreated control group, which received 5% dextrose solution iv. Therapy with AMB (1.5 mg/kg/day, ip) and LAMB (10 mg/kg/day, iv) began 1 day after infection and lasted for 10 days. AMB and LAMB were administered by different routes because AMB is toxic for mice when given iv at doses higher than 1 mg/kg/day.7 Therapy with G-CSF (300 µg/kg/day, ip) began 3 days before infection and lasted for 8 days (from day -3 to day +5). Mortality was recorded daily for 30 days.
For the tissue burden study three groups of 10 animals were infected with 0.2 mL of S. prolificans at 2.5 x 104 conidia/mL, which were treated with LAMB + G-CSF, AMB or 5% glucose, respectively. The treatment regimes were identical to those in the survival study. On day 11 after challenge the surviving mice (approximately four to six per group) were anaesthetized with halothane (Fluothane; Zeneca Farma, S.A., Pontevedra, Spain) and humanely killed by cervical dislocation. The brain, lungs and kidneys were aseptically removed, and half of each organ was weighed and homogenized in 1 mL of sterile saline solution. This produced a minimal threshold of c. 210 cfu/ organ. Six 10-fold serial dilutions of this homogenate were made with sterile saline. Volumes of 0.1 and 0.5 mL of the homogenate and 0.1 mL of each dilution were inoculated on to plates of SDA and incubated at 30°C for 3 days. Colonies were counted and plates with <200 colonies were used. The other halves of the organs were fixed in 10% neutral buffered formaldehyde for 10 days, embedded in paraffin wax and automatically processed. Sections (3 µm in thickness) of the embedded tissues were stained with haematoxylineosin, periodic acid Schiff (PAS) and methenamine silver (Grocott) for light microscopy observations.
Statistics
Mean survival time (MST) was estimated by the Kaplan Meier method and compared among groups using the log-rank test. Colony counts in tissue burden studies were analysed by a parametric method that consisted of an analysis of variance. Calculations were performed using SPSS for Windows version 9.0.
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Results |
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Discussion |
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The combination of LAMB and G-CSF was clearly the most effective treatment. This therapy regime allowed the longest MST and the best survival rate. The combination of these two agents could provide important advantages for the treatment of refractory infections. The lipid vehicle of LAMB reduces toxicity and allows administration of higher doses, which results in higher concentrations of AMB in serum than treatment with conventional AMB.7 In addition, a more optimal tissue distribution is reached by LAMB, resulting in a higher concentration of the drug in reticuloendothelial tissues heavily involved in fungal infections.10 Clinical studies have demonstrated the efficacy of this compound for empirical antifungal therapy in neutropenic patients.1113 G-CSF could contribute to recovery from the infection by increasing the number of differentiated granulocytes and their phagocytic activity.14,15 Our study seems to confirm this because histological examination revealed a greater inflammatory response by PMNs and a lower number of fungal cells in the LAMB + G-CSF-treated group than in the other groups. Graybill et al.16 reported no effect of G-CSF alone on survival of experimental cryptococcal meningitis, in agreement with Clemons et al.17 in their study of orogastrointestinal candidosis. However, the combination of G-CSF with antifungal drugs showed an additive effect in experimental murine aspergillosis.8
Treatment with AMB was ineffective in our study. In contrast, treatment with LAMB alone improved the MST compared with the control group, although all the animals died before the end of the testing. This latter compound has been used in several human infections caused by S. prolificans and in one of only two cases of disseminated infection that have been resolved.4,5 In a previous trial (data not published) the strain used in this study clearly showed in vitro resistance to AMB (MIC > 16 mg/L) by the method adapted from the National Committee for Clinical Laboratory Standards for moulds.18 This agrees with other in vitro studies that have shown high resistance of S. prolificans to AMB. 4,6
Our experimental results, although still preliminary, are promising for future treatment of S. prolificans infections in neutropenic patients. It is even possible that by increasing the LAMB doses and/or making the cytokine regimen more suitable, better results can be obtained. Some of the new antifungals are also promising candidates for new therapeutic strategies. For instance, the new triazole UR-9825 (Uriach, Barcelona, Spain) has showed excellent in vitro results.2 Synergy of several antifungal drugs against clinical isolates of this species has also been documented in vitro.19,20 Nevertheless, these findings need to be viewed with caution and before using these compounds in the clinical setting, proof of their efficacy in adequate animal models is required.
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Acknowledgements |
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Notes |
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References |
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20 . Pacheco, P., Sutton, D., Fothergill, A. & Rinaldi, M. (2000). In vitro susceptibility of Scedosporium prolificans human isolates to several drugs used alone and in combination. In Program and Abstracts of the Fourteenth Congress of the International Society of Human and Animal Mycology. Abstract 79, p. 234. ISHAM, Buenos Aires, Argentina.
Received 15 January 2001; returned 20 June 2001; revised 25 September 2001; accepted 27 November 2001