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.
Mean survival time was estimated by a Kaplan-Meier method and compared
among groups by using a log rank test. Colony counts in tissue burden studies
were analyzed by a Kruskal-Wallis test. When this test was significant, we used a
Mann-Whitney U test to compare pairs of strains. The Bonferroni correction was
used to avoid an increase of the type I error due to multiple comparisons.
Differences were considered statistically significant at a P value of ?0.05.
The in vitro susceptibility test showed AMB and VRC MICs
of 0.5 mg/liter and a PSC MIC of 0.25 mg/liter for strains FMR
8394 and FMR 8396 while for strain FMR 8410 MICs were
0.12 mg/liter, 0.12 mg/liter, and 0.25 mg/liter, respectively.
Results of survival studies are shown in Fig. 1. AMB and
PSC significantly prolonged survival with respect to survival of
the control group for all the strains, with the exception of PSC
at 10 and 20 mg/kg against strain FMR 8410. In addition, AMB
was able to significantly prolong survival relative to survival
with the rest of the therapies for strains FMR 8410 and FMR
8394 and relative to survival with VRC against strain FMR
8396. None of the VRC doses prolonged survival, with the
exception of VRC at 60 mg/kg against strain FMR 8396.
The tissue burden results are shown in Tables 1 and 2. On
day 5, for all strains all treatments, except VRC at 10 mg/kg,
significantly reduced the fungal load in lung and spleen relative
to the load in the control group. In addition, AMB significantly
reduced the fungal load in these organs in comparison to VRC
at any dose for strains FMR 8396 and 8410 and to PSC at 10
and 20 mg/kg for all strains, with the exception of the lung
against strain FMR 8396. AMB showed better efficacy than
PSC at 40 mg/kg in reducing the fungal load only in the spleen
against strain FMR 8396 and in lungs against strain FMR 8410.
Overall, PSC showed significantly better efficacy than VRC in
reducing the fungal recovery in spleen, and both drugs had
similar activities in lungs. None of the treatments reduced the
fungal load in brain against the strain FMR 8396. Against
strains FMR 8394 and FMR 8410, AMB and PSC at 20 and 40
mg/kg reduced the tissue burden in brain in comparison to the
burden in the control group. Against strain FMR 8394, PSC at
40 mg/kg significantly reduced the brain fungal load relative to
the load with AMB.
On day 11, AMB cleared the fungal load in spleen and lungs
and significantly reduced the tissue burden in brain in compar-
ison to the results of day 5. Against strain FMR 8394, all PSC
doses cleared fungal loads in spleen and lungs, and PSC at 40
mg/kg cleared the fungal load in spleen against strain FMR
8396. In the brain, all groups treated with VRC and PSC
surprisingly showed higher fungal loads than at day 5.
Bioassay results are shown in Table 3. At day 10 of therapy,
for all treatments administered, antifungal levels in serum and
brain were above the corresponding MICs. PSC and VRC
levels in serum and brain increased with dose escalation.
We have studied the efficacy of PSC, VRC, and AMB in the
treatment of a disseminated infection by C. gattii in immuno-
suppressed mice. Although C. gattii seems to have a predilec-
tion for immunocompetent hosts, it is also known to infect
immunocompromised patients such as those with AIDS (2, 16,
17, 25). It has been suggested that the differences in host
incidence between Cryptococcus species could be related to a
lesser exposure of AIDS patients to C. gattii since this species
FIG. 1. The cumulative mortality of mice infected with 2 ? 105CFU of C. gattii. Significance is indicated on the figure as follows: a, P ? 0.05
versus control; b, P ? 0.05 versus PSC at 10 mg/kg; c, P ? 0.05 versus PSC at 20 mg/kg; d, P ? 0.05 versus VRC at 10 mg/kg; e, P ? 0.05 versus
VRC at 40 mg/kg; f, P ? 0.05 versus VRC at 60 mg/kg; and g, P ? 0.05 versus all the therapies.
VOL. 54, 2010ANTIFUNGAL THERAPY AGAINST CRYPTOCOCCUS GATTII4075
is less common in the urban areas where these patients typi-
cally reside (6).
PSC and VRC have shown efficacy in human infections by C.
neoformans (9, 15, 21, 22), but little data exist on their efficacy
against C. gattii infections. To our knowledge, this is the first
study that explores the efficacy of these drugs in a murine C.
gattii infection. In contrast to the high in vitro activities of PSC
and VRC, which agree with the findings of other authors (13,
29), these drugs showed lower efficacy than AMB in vivo
against C. gattii. In this study, the high mortality of mice treated
with VRC or PSC seems to be associated with the inability of
these drugs to reduce the fungal load in the brain despite
increased drug levels in the organ. In contrast, these drugs,
especially PSC, showed efficacy in reducing the fungal load in
lung. These poor results obtained with PSC and VRC in brain
tissue could be related to their fungistatic activity and to the
TABLE 1. The effect of antifungal treatment on colony counts in lung, spleen, and brain on day 5 of therapy
Isolate Drug (dose ?mg/kg/day?)
Mean log10CFU/g (95% CI)h
Lung Spleen Brain
aP ? 0.005 versus control.
bP ? 0.005 versus VRC at 10 mg/kg.
cP ? 0.005 versus VRC 40 at mg/kg.
dP ? 0.005 versus VRC at 60 mg/kg.
eP ? 0.005 versus all the therapies.
fP ? 0.005 versus PSC at 10 mg/kg.
gP ? 0.005 versus PSC at 20 mg/kg.
hCI, confidence interval; ND, not detected.
TABLE 2. The effect of antifungal treatment on colony counts in lung, spleen, and brain on day 11 after challenge
IsolateDrug (dose ?mg/kg/day?)
Mean log10CFU/g (95% CI)c
0.87 (?0.10 to 1.84)a
aP ? 0.05 versus fungal load on day 5 of therapy (significantly lower).
bP ? 0.05 versus fungal load on day 5 of therapy (significantly higher).
cCI, confidence interval; —, control mice did not survive to day 11; ND, not detected.
4076CALVO ET AL.ANTIMICROB. AGENTS CHEMOTHER.
inhibition of the lymphocyte function provoked by cyclophos-
phamide administration (12, 18). It has been suggested that a
T-cell-independent defense mechanism in pulmonary C. neo-
formans infections (14) together with the antifungal adminis-
tration could explain the fungal clearance in that organ in our
model. It is likely that such a defensive response would not be
expressed in brain (14). T cells are necessary to reduce the
cryptococcal burden, and their depletion would provoke an
increase in the brain fungal load (4, 30).
In summary, the poor efficacy of PSC and VRC in reducing
the fungal load in brain and in prolonging mouse survival in an
acute disseminated cryptococcosis caused by C. gattii suggests
that, as other azoles, these drugs should not be administered as
primary therapies. PSC and VRC could be alternatives to itra-
conazole and fluconazole in the maintenance of therapy and,
as was observed here, in the treatment of pulmonary crypto-
1. Barchiesi, F., A. M. Schimizzi, F. Caselli, D. Giannini, V. Camiletti, B.
Fileni, A. Giacometti, L. F. Di Francesco, and G. Scalise. 2001. Activity of
the new antifungal triazole, posaconazole, against Cryptococcus neoformans.
J. Antimicrob. Chemother. 48:769–773.
2. Bodasing, N., R. A. Seaton, G. S. Shankland, and D. Kennedy. 2004. Cryp-
tococcus neoformans var. gattii meningitis in an HIV-positive patient: first
observation in the United Kingdom. J. Infect. 49:253–255.
3. Bodet C. A., III, J. H. Jorgensen, and D. J. Drutz. 1985. Simplified bioassay
method for measurement of flucytosine or ketoconazole. J. Clin. Microbiol.
4. Buchanan, K. L., and H. A. Doyle. 2000. Requirement for CD4?T lympho-
cytes in host resistance against Cryptococcus neoformans in the central ner-
vous system of immunized mice. Infect. Immun. 68:456–462.
5. Reference deleted.
6. Chen, Y. C., S. C. Chang, C. C. Shih, C. C. Hung, K. T. Luhbd, Y. S. Pan, and
W. C. Hsieh. 2000. Clinical features and in vitro susceptibilities of the two
varieties of Cryptococcus neoformans in Taiwan. Diagn. Microbiol. Infect.
7. Christiansen, K. J., E. M. Bernard, J. W. Gold, and D. Armstrong. 1985.
Distribution and activity of amphotericin B in humans. J. Infect. Dis. 152:
8. Clinical and Laboratory Standards Institute. 2008. Reference method for
broth dilution antifungal susceptibility of yeasts. Approved standard M27-
A3, 3rd ed. Clinical and Laboratory Standards Institute, Wayne, PA.
9. Esposito, V., R. Viglietti, M. Gargiulo, R. Parrella, M. Onofrio, V. Sangio-
vanni, D. Ambrosino, and A. Chirianni. 2009. Successful treatment of cryp-
tococcal meningitis with a combination of liposomal amphotericin B, flucy-
tosine and posaconazole: two cases reports. In Vivo 23:465–468.
10. Fittler, A., B. Kocsis, I. Gerlinger, and L. Botz. 2010. Optimization of
bioassay method for the quantitative microbiological determination of am-
photericin B. Mycoses 53:57–61.
11. Friese, G., T. Discher, R. Fu ¨ssle, A. Schmalreck, and J. Lohmeyer. 2001.
Development of azole resistance during fluconazole maintenance therapy for
AIDS-associated cryptococcal disease. AIDS 15:2344–2345.
12. Glu ¨ck, T., B. Kiefmann, M. Grohmann, W. Falk, R. H. Straub, and J.
Scho ¨lmerich. 2005. Immune status and risk for infection in patients receiving
chronic immunosuppressive therapy. J. Rheumatol. 32:1473–1480.
13. Go ´mez-Lo ´pez, A., O. Zaragoza, M. Dos Anjos Martins, M. C. Melhem, J. L.
Rodríguez-Tudela, and M. Cuenca-Estrella. 2008. In vitro susceptibility of
Cryptococcus gattii clinical isolates. Clin. Microbiol. Infect. 14:727–730.
14. Hill, J. O., and P. L. Dunn. 1993. A T cell-independent protective host
response against Cryptococcus neoformans expressed at the primary site of
infection in the lung. Infect. Immun. 61:5302–5308.
15. Izumikawa, K., Y. Zhao, K. Motoshima, T. Takazono, T. Saijo, S. Kurihara,
S. Nakamura, T. Miyazaki, M. Seki, H. Kakeya, Y. Yamamoto, K. Yanagi-
hara, Y. Miyazaki, T. Hayashi, and S. Kohno. 2008. A case of pulmonary
cryptococcosis followed by pleuritis in an apparently immunocompetent pa-
tient during fluconazole treatment. Med. Mycol. 46:596–599.
16. Lindenberg Ade, S., M. R. Chang, A. M. Paniago, Mdos S. La ´zera, P. M.
Moncada, G. F. Bonfim, S. A. Nogueira, and B. Wanke. 2008. Clinical and
epidemiological features of 123 cases of cryptococcosis in Mato Grosso do
Sul, Brazil. Rev. Inst. Med. Trop. Sao Paulo 50:75–78.
17. Litvintseva, A. P., R. Thakur, L. B. Reller, and T. G. Mitchell. 2005. Prev-
alence of clinical isolates of Cryptococcus gattii serotype C among patients
with AIDS in sub-Saharan Africa. J. Infect. Dis. 192:888–892.
18. Lutsiak, M. E., R. T. Semnani, R. De Pascalis, S. V. Kashmiri, J. Schlom,
and H. Sabzevari. 2005. Inhibition of CD4?25?T regulatory cell function
implicated in enhanced immune response by low-dose cyclophosphamide.
19. Perfect, J. R., G. M. Cox, R. K. Dodge, and W. A. Schell. 1996. In vitro and
in vivo efficacies of the azole SCH56592 against Cryptococcus neoformans.
Antimicrob. Agents Chemother. 40:1910–1913.
20. Perfect, J. R., W. E. Dismukes, F. Dromer, D. L. Goldman, J. R. Graybill,
R. J. Hamill, T. S. Harrison, R. A. Larsen, O. Lorthoraly, M. H. Nguyen,
P. G. Pappas, W. G. Powderly, N. Singh, J. D. Sobel, and T. C. Sorrell. 2010.
Practice guidelines for the management of cryptococcal disease: 2010 update
by the infectious diseases society of America. Clin. Infect. Dis. 50:291–322.
21. Perfect, J. R., K. A. Marr, T. J. Walsh, R. N. Greenberg, B. DuPont, J. de la
Torre-Cisneros, G. Just-Nu ¨bling, H. T. Schlamm, I. Lutsar, A. Espinel-
Ingroff, and E. Johnson. 2003. Voriconazole treatment for less-common,
emerging, or refractory fungal infections. Clin. Infect. Dis. 36:1122–1131.
22. Pitisuttithum, P., R. Negroni, J. R. Graybill, B. Bustamante, P. Pappas, S.
Chapman, R. S. Hare, and C. J. Hardalo. 2005. Activity of posaconazole in
the treatment of central nervous system fungal infections. J. Antimicrob.
23. Saag, M. S., J. R. Graybill, R. A. Larsen, P. G. Pappas, J. R. Perfect, W. G.
Powderly, J. D. Sobel, and W. E. Dismukes. 2000. Practice guidelines for the
management of cryptococcal disease. Clin. Infect. Dis. 30:710–718.
24. Serena, C., F. J. Pastor, M. Marine ´, M. Mar Rodríguez, and Josep Guarro.
2007. Efficacy of voriconazole in a murine model of cryptococcal central
nervous system infection. J. Antimicrob. Chemother. 60:162–165.
25. Severo, L. C., F. de Mattos Oliveira, and A. T. Londero. 1999. Cryptococcosis
due to Cryptococcus neoformans var. gattii in brazilian patients with AIDS.
Report of three cases. Rev. Iberoam. Micol. 16:152–154.
26. Soares, B. M., D. A. Santos, L. M. Kohler, G. da Costa Ce ´sar, I. R. de
Carvalho, M. dos Anjos Martins, and P. S. Cisalpino. 2008. Cerebral infec-
tion caused by Cryptococcus gattii: a case report and antifungal susceptibility
testing. Rev. Iberoam. Micol. 31:242–245.
27. Sorrell, T. C. 2001. Cryptococcus neoformans variety gattii. Med. Mycol.
28. Sugar, A. M., and X. Liu. 2001. Efficacy of voriconazole in treatment of
murine pulmonary blastomycosis. Antimicrob. Agents Chemother. 45:601–
29. Thompson, G. R., III, N. P. Wiederhold, A. W. Fothergill, A. C. Vallor, B. L.
Wickes, and T. F. Patterson. 2009. Antifungal susceptibilities among differ-
ent serotypes of Cryptococcus gattii and Cryptococcus neoformans. Antimi-
crob. Agents Chemother. 53:309–311.
30. Uicker, W. C., J. P. McCracken, and K. L. Buchanan. 2006. Role of Cd4?T
cells in a protective immune response against Cryptococcus neoformans in the
central nervous system. Med. Mycol. 44:1–11.
TABLE 3. The drug levels in serum and brain tissue measured by
bioassay on day 10 of the therapy and 4 h after final dosinga
Drug and dose (mg/kg) Serum (?g/ml)Brain (?g/g)
5.75 ? 1.86
6.73 ? 1.29
8.83 ? 0.79
3.08 ? 0.36
3.64 ? 0.91
7.41 ? 1.12
4.09 ? 0.98
5.54 ? 1.55
7.16 ? 2.13
2.62 ? 0.42
4.12 ? 0.97
6.47 ? 1.03
1.56.52 ? 1.474.46 ? 0.73
aResults are expressed as the means ? standard deviations.
VOL. 54, 2010ANTIFUNGAL THERAPY AGAINST CRYPTOCOCCUS GATTII4077