a Department of Medicine, Division of Infectious Diseases, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, Mail Code 7881, San Antonio, TX 78229-3900; b Audie Murphy Division, South Texas Veterans Health Care System, San Antonio, TX 78284, USA
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Abstract |
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Introduction |
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We used a guinea pig model of invasive aspergillosis to assess the effectiveness of antifungal therapy in this disseminated disease.5,15 In this lethal model, guinea pigs were made leucopenic and additional immune-system suppression was induced with concomitant steroid administration. The development of pervasive infection in the liver, kidney, lung and brain was comparable to clinically disseminated invasive aspergillosis.16,17 We evaluated the activity of ravuconazole in experimental invasive aspergillosis and compared its efficacy with that of amphotericin B and itraconazole.
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Materials and methods |
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Male Hartley guinea pigs (0.5 kg) were immunosuppressed with triamcinolone acetonide 20 mg/kg sc od (Steris Laboratories, Phoenix, AZ, USA) beginning 4 days before challenge and rendered neutropenic with cyclophosphamide 300 mg/kg ip (Pharmacia, Kalamazoo, MI, USA) 1 day before challenge. With this temporarily immunosuppressive regimen, the guinea pigs had reduced total white blood cell counts through day 7. Twenty-four hours after induction of neutropenia, groups of eight to 10 guinea pigs were sedated with ketamine HCl 44 mg/kg (Fort Dodge Laboratories, Fort Dodge, IA, USA), atropine 0.04 mg/kg (Elkin-Sinn, Cherry Hill, NJ, USA) and xylazine 5 mg/kg (Bayer Corporation Agriculture Division, Shawnee Mission, KS, USA), and challenged intravenously through the saphenous vein with a lethal inoculum of 106 A. fumigatus conidia. Each group contained at least one untreated control guinea pig, for which the lethal challenge was fatal within 6 days, with a mean survival of 4.8 ± 0.4 days (range 36 days) after challenge. The administration of ceftazidime 100 mg/kg im od (SmithKline Beecham Pharmaceuticals, Philadelphia, PA, USA) began on the day of challenge to prevent intercurrent bacterial infection. All animal research procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Center (San Antonio, TX, USA).
Antifungal agents
Antifungal treatment comprised amphotericin B (Fungizone; Bristol-Myers Squibb Co., Princeton, NJ, USA), ravuconazole (Bristol-Myers Squibb Co.) or itraconazole cyclodextrin solution (Janssen Research Foundation, Beerse, Belgium), and began 24 h after challenge with A. fumigatus conidia and continued for 5 days. Amphotericin B was diluted with 5% dextrose in sterile water at a ratio of 1.25 mg/mL of diluent and was administered at 1.25 mg/kg ip od. Ravuconazole was suspended in polyethylene glycol (PEG) 400 (Sigma Chemical, St Louis, MO, USA) and administered as a 10 mg/mL suspension at 5, 10 or 25 mg/kg po od. Itraconazole (10 mg/mL) was administered at 2.5 or 5.0 mg/kg po bd for a total dose of 5 mg/ kg/day or 10 mg/kg/day, respectively.
Organ cultures
Organ cultures were carried out post-mortem [after the death of the animal during treatment (n = 12) or 96 h after completion of treatment in the remaining treated guinea pigs (n = 44)]. Guinea pigs were humanely killed by terminal exsanguination after being anaesthetized with ketamine HCl 44 mg/kg and xylazine 10 mg/kg. Organs (brain, lung, liver and kidneys) were removed aseptically and were cultured to determine the degree of infection with A. fumigatus. Organs were considered positive when three or more colonies of A. fumigatus were present on minced tissues placed directly on Sabouraud Dextrose Agar (SDA) plates (Becton Dickinson and Co., Cockeysville, MD, USA) or when semi-quantitative cultures of tissue homogenates contained over 30 cfu/g of tissue.5 Tissue burdens of Aspergillus were evaluated with semi-quantitative cultures that could detect 3020 000 cfu/g of tissue.18 Samples of each organ were finely chopped (manually), weighed, diluted 1:10 (w/v) with sterile saline and homogenized for 25 s with an electric tissue homogenizer (IKA-Works, Cincinnati, OH, USA). Duplicate 0.1 and 1.0 mL samples of the organ homogenate were plated on SDA plates, incubated at 37°C for 48 h, and colonies were counted. In combination, these two methods detected A. fumigatus at 320 000 cfu/g of tissue.
Organisms
A. fumigatus isolate P171, a clinical isolate that had been used in previous animal studies, was grown on SDA slants at 37°C for 24 h. The 24/48 h MICs of ravuconazole for this isolate were 0.25/1 mg/L. For injection into the guinea pigs, conidia were harvested by a sterile saline wash of the slant surface, with conidia being dislodged by gentle rubbing with a sterile glass rod. The resultant conidia suspension was adjusted to the desired concentration of 106 conidia/mL by haemocytometer count, which was verified by duplicate serial plating on SDA plates for colony counts.
Statistical analysis
The Fisher exact test and the Wilcoxon rank sum test were used where appropriate. Statistical significance was defined as P < 0.05, but adjustments were made for multiple dose comparisons for each organ evaluated so that the level of significance was P < 0.003.
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Results |
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Discussion |
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Acknowledgements |
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Notes |
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References |
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Received 14 May 2001; returned 17 July 2001; revised 23 August 2001; accepted 31 October 2001