ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2010, p. 4074–4077
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 10
Antifungal Therapy in a Murine Model of Disseminated
Infection by Cryptococcus gattii?
Enrique Calvo,1F. Javier Pastor,1M. Mar Rodríguez,1Isabel Pujol,2and Josep Guarro1*
Unitat de Microbiologia, Facultat de Medicina, IISPV, Universitat Rovira i Virgili, Reus, Spain,1and
Laboratori de Microbiologia, Hospital Universitari Sant Joan de Reus, Reus, Spain2
Received 5 February 2010/Returned for modification 28 April 2010/Accepted 3 July 2010
We have evaluated the efficacy of posaconazole (PSC), voriconazole (VRC), and amphotericin B (AMB) in
a murine model of systemic infection by Cryptococcus gattii using immunocompromised animals and three
clinical strains of the fungus. AMB was the most effective drug in prolonging the survival of mice and also in
reducing tissue burden in all organs tested. To a lesser degree, VRC at 60 mg/kg of body weight in lung tissue
and PSC at 40 mg/kg also in spleen demonstrated good efficacy in reducing the fungal load. The PSC and VRC
levels in serum and brain tissue, determined by an agar diffusion bioassay method at 4 h after the last dose of
the therapy, were above the corresponding MIC values. However, these drugs were not able to reduce the fungal
load in brain tissue. Our results demonstrated that PSC and, to a lesser degree, VRC, have fungistatic activity
and potential for the treatment of human pulmonary cryptococcosis.
Cryptococcosis is an emerging infection commonly involving
the lungs, from which it can disseminate to different tissues,
usually the central nervous system (CNS) (20, 23). Cryptococ-
cus neoformans and Cryptococcus gattii are the main agents
responsible for this disease, which can affect both immunosup-
pressed and healthy individuals. Despite antifungal therapies,
this infection still has mortality rates near 20% (20).
The first choice in the primary therapy of CNS infections
remains fungicidal drugs, with amphotericin B (AMB) alone or
in combination with flucytosine being the most widely used (20,
23). Fungistatic drugs like itraconazole and fluconazole, with
less toxicity, are also used in the maintenance of the therapy
and in pulmonary cryptococcosis, but their use in CNS infec-
tions has been less than satisfactory. In addition, the extended
duration of the therapy with these azoles increases the risk of
developing drug resistance (11, 23, 26). It has been suggested
that that C. gattii has a higher pathogenicity than C. neofor-
mans (27), which emphasizes the importance of the correct
species identification and makes it necessary to improve and
search for alternatives to the current therapy.
On the basis of the promising results obtained with posacon-
azole (PSC) and voriconazole (VRC) against C. neoformans in
animal models (1, 19, 24) and also in a clinical setting (9, 15, 21,
22), we have evaluated in this study the efficacy of PSC, VRC,
and AMB in a murine model of disseminated infection by C.
MATERIALS AND METHODS
Three clinical isolates of C. gattii, FMR 8394, FMR 8396, and FMR 8410, were
used in this study. We tested their in vitro antifungal susceptibilities to AMB,
PSC, and VRC by using a broth microdilution method following the CLSI
guidelines for yeasts (8).
In the in vivo study, male OF1 mice (Charles River, Criffa S.A., Barcelona,
Spain) with a mean weight of 30 g were used. The 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.
Mice were immunosuppressed by a single intraperitoneal (i.p.) injection of 200
mg/kg of body weight of cyclophosphamide (Genoxal; Laboratorios Funk S.A.,
Barcelona, Spain) plus 5-fluorouracil (Fluorouracilo; Ferrer Farma S.A., Barce-
lona, Spain) at 150 mg/kg intravenously 1 day before the infection. In order to
prevent bacterial infections, all mice received 5 mg/day ceftazidime subcutane-
ously from days 1 to 7 after the infection.
On the day of infection, 1-day cultures on potato dextrose agar (PDA) were
suspended in sterile saline and filtered through sterile gauze to remove clumps of
cells. The resulting suspension was adjusted to the desired inoculum based on
hemocytometer counts. Dilutions of the original suspension were cultured on
PDA plates to confirm the hemocytometer counts. Mice were infected with a
conidial suspension of 2 ? 105CFU in 0.2 ml of sterile saline solution into the
lateral tail vein. This inoculum was chosen in previous studies and was the
minimal dose able to produce acute infections, with all the animals dying within
15 days postinfection (data not shown).
The drugs assayed were AMB (Fungizone; Squibb Industrial Farmace ´utica
S. A., Barcelona, Spain), PSC (Noxafil; Schering-Plough Ltd., Hertfordshire,
United Kingdom), and VRC (Vfend; Pfizer S.A., Madrid, Spain), administered
as follows: AMB at doses of 1.5 mg/kg of body weight, i.p. once daily; PSC at
doses of 10, 20, or 40 mg/kg orally once daily; or VRC at doses of 10, 40, or 60
mg/kg orally once daily. From 3 days before infection, the mice that received
VRC were given grapefruit juice instead of water (28). Control animals received
no treatment. All treatments began 1 day after challenge, and the therapy lasted
for 10 days. The efficacy of the different drugs was evaluated by prolongation of
survival and reduction of fungal tissue burden.
Groups of 30 mice were randomly established for each strain and treatment.
Ten mice were used for survival studies, and 20 mice were used for tissue burden
studies. For each strain and study, groups of 10 mice were also established as
controls. Mice in the survival group were checked daily for 30 days. At the end
of the experiment, survivors were sacrificed by carbon dioxide inhalation. The
mice in one tissue burden group were sacrificed on day 5, and the other mice
were sacrificed on day 11 postinfection; their lungs, brain, and spleen were
aseptically removed and homogenized in 1 ml of sterile saline. Serial 10-fold
dilutions of the homogenates were plated on PDA, incubated at 30°C, and
examined daily for 3 days. The number of CFU/g of tissue was calculated for each
An additional group of five mice was similarly infected with the strain FMR
8396 and treated with the same antifungals and doses used in the treatment
study. These mice were used to determine antifungal drug levels in the brain and
serum 4 h after the final dose on day 10 of therapy. Levels were determined by
bioassay following described methods for quantification of azoles and AMB in
serum and tissues (3, 7, 10).
* Corresponding author. Mailing address: Unitat de Microbiologia,
Facultat de Medicina, Universitat Rovira i Virgili. Carrer Sant
Llorenc ¸, 21.43201 Reus, Spain. Phone: 977 759359. Fax: 977 759322.
?Published ahead of print on 12 July 2010.