Mycopathologia (2012) 173:445449 DOI 10.

1007/s11046-011-9499-2

Voriconazole, Combined with Amphotericin B, in the Treatment for Pulmonary Cryptococcosis Caused by C. neoformans (Serotype A) in Mice with Severe Combined Immunodeciency (SCID)
Eriques Gonc alves Silva Claudete Rodrigues Paula Francisco de Assis Baroni Walderez Gambale

Received: 6 June 2011 / Accepted: 28 October 2011 / Published online: 10 November 2011 Springer Science+Business Media B.V. 2011

Abstract Cryptococcosis is a subacute or chronic systemic mycosis with a cosmopolitan nature, caused by yeast of the genus Cryptococcus neoformans. The model of systemic cryptococcosis in mice with severe combined immunodeciency (SCID) is useful for immunological and therapeutic study of the disease in immunodecient hosts. Amphotericin B, uconazole and ucytosine are the drugs most commonly used to treat cryptococcosis. Voriconazole is a triazole with high bioavailability, large distribution volume, and excellent penetration of the central nervous system. The objective of this study was to evaluate treatment with amphotericin B (AMB), voriconazole (VRC), and AMB, used in combination with VRC, of experimental pulmonary cryptococcosis in a murine model (SCID). The animals were inoculated intravenously (iv) with a solution containing 3.0 9 105 viable cells of C. neoformans ATCC 90112, (serotype A). Treatments were performed with amphotericin B (1.5 mg/ kg/day), voriconazole (40.0 mg/kg/day) and AMB (1.5 mg/kg/day) combined with VRC (40.0 mg/kg/ day); began 1 day after the initial infection; were daily;
E. G. Silva (&) C. R. Paula W. Gambale Department of Microbiology, Institute of Biomedical o Paulo (USP), Sciences, University of Sa o Paulo, SP, Brazil Sa e-mail: eriques@usp.br F. de Assis Baroni Federal Rural University of Rio de Janeiro (UFRRJ), dica, RJ, Brazil Serope

and lasted 15 days. Evaluations were performed using analysis of the survival curve and isolation of yeast in the lung tissue. There was a signicant increase in survival in groups treated with AMB combined with VRC, compared with the untreated group and groups receiving other treatments (P \ 0.05). In the group treated only with VRC and AMB combined with VRC, there was a signicant reduction (P \ 0.05) in the isolation of C. neoformans in lung tissue. Amphotericin B combined with voriconazole may be an effective alternative to increasing survival and may reduce yeast in the lung tissue of mice with pulmonary cryptococcosis and SCID. Keywords Amphotericin B Voriconazole Combination therapy Cryptococcus neoformans SCID Severe combined immunodeciency

Introduction The Cryptococcus neoformans variety of neoformans is a pathogenic opportunistic yeast, commonly found in the excreta of pigeons and other birds [1]. The yeast is responsible for the subacute or chronic, systemic mycoses cryptococcosis [2]. Cryptococcosis diseases are caused through the inhalation of infective particles of yeast, which stay in the lungs, causing pulmonary cryptococcosis [2]. Through the bloodstream, C. neoformans arrives at other organs, mainly in the central nervous system (CNS) [3]. The murine model of Severe

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Combined Immunodeciency (SCID) is more susceptible to systemic experimental cryptococcosis and can be a good model for the study of immunology and therapeutics [4]. Its susceptibility is greater because B and T cells, which protect against severe disease, are not expressed [5]. Amphotericin B, uconazole, and uorocytosine are the drugs most used in cryptococcosis treatment therapies [6]. For more than 30 years, amphotericin B has been used to treat severe fungal infections, but its use has been limited due to various adverse effects, the most important being nephrotoxicity [7, 8]. Fluconazole is the drug most often used to treat cerebral cryptococcosis [9], but indiscriminate use over long periods can result in resistance [10]. Voriconazole is a triazole with high bioavailability, large distribution volume, and excellent penetration of the CNS [11]. It also shows excellent activity in vitro against C. neoformans [12], achieving good concentration levels in the serum [9]. Antifungal azoles, combined with other antifungal agents that have differing mechanisms of action, increase the efciency of treatments for diseases caused by fungal infections [13], reducing the development of resistance to these drugs and, possibly, decreasing length of treatment [14]. The objective of this study was to evaluate treatment with amphotericin B voriconazole alone, and amphotericin B combined with voriconazole, of experimental pulmonary infections caused by C. neoformans in a murine model with Severe Combinated Immunodeciency (SCID).

mean weight of 20 g were obtained from the Animal Center, which is responsible for breeding isogenic animals at the Institute for Energy and Nuclear o Paulo, Brazil. These mice were Research, in Sa housed in microisolator cages, provided with sterile feed and water, and randomly distributed into ve groups. The C. neoformans ATCC 90112 was cultivated in a YPD medium of 1% yeast extract (Difco), 1% Bacto Peptone (Difco), and 2% dextrose (SigmaAldrich, Milwaukee, WI, USA) for 18 h at 30C; the cells were collected after centrifugation, washed twice in a phosphate buffer solution (PBS), and resuspended at the inoculation concentration. Four groups were inoculated intravenously with 100 lL of the suspension, containing 3.0 9 105 viable cells of the yeast. Among these four groups, 3 groups (with 5 animals each) were treated, one group (n = 10) was not treated and served as the positive control, and one group (n = 10) was inoculated with PBS and served as the negative control. Animal handling and treatment were conducted in accordance with the Ethical Principals of the Brazilian College of Animal Experimentation (COBEA). Treatments Treatments began 1 day after the initial inoculation, were daily, and lasted for 15 days. The animals were inoculated intraperitoneally with 0.1 mL of amphotericin B (1.5 mg/kg/day) (Fungizone, Bristol-Meyers, Squibb S.p.A., Sermoneta, Italy) [13], 0.1 mL of voriconazole (40.0 mg/kg/day) (Vfend IV) (Pzer Inc, New York, NY, USA) [14, 15], and 0.1 mL of amphotericin B (1.5 mg/kg/day), in combination with 0.1 mL of voriconazole (40.0 mg/kg/ day). At the end of the study period (50 days), all animals that survived were euthanized in a CO2 chamber. The survival curves and yeast content of the lung tissue (CFU) of dead and surviving mice were evaluated. Removal of Lungs Lungs were aseptically removed, weighed, and homogenized in 2 mL of sterile saline solution. Quantication of Yeast in Lungs (CFUs) Ten-fold serial dilutions of the tissue homogenates were plated (100 lL) in triplicate on SDA (Difco),

Materials and Methods Cryptococcus neoformans The study was performed using the C. neoformans ATCC 90112 (serotype A). This strain was maintained in tubes containing Sabouraud dextrose agar (Difco Laboratories, Detroit, MI, USA) and glycerol at 20C, in the Laboratory of Pathogenic Yeasts of the Department of Microbiology, at the Institute of o Paulo University, Sa o Biomedical Sciences at Sa Paulo, Brazil. Animal Models and Experimental Cryptococcosis Were used in the study 35 murine models with Severe Combinated Immunodeciency (SCID), with and a

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incubated at 35C, and examined daily for 3 days. The number of CFU per gram of tissue was calculated. Statistical Analysis Mean survival times were estimated using the Kaplan Meier method and compared among groups, using the log-rank test. The data obtained in relation to the CFUs in lung homogenates underwent logarithmic transformation, to achieve an approximation of normal distribution, prior to statistical analysis using the MannWhitney test. All statistical tests were performed using the software GraphPad Prisma 5 (GraphPad PrismTM, Version 5.0, and GraphPad Software Incorporated). Differences were considered signicant when P \ 0.05.

Discussion This studys model of cryptococcosis in mice with Severe Combined immunodeciency (SCID) can be useful in the immunologic and therapeutic study of diseases in hosts with immunodeciency [4]. Amphotericin B has been successfully used for over 30 years in the treatment for cryptococcosis, whose toxicity, in addition to an increase in strains resistant to
100 80
Control AMB a, c VRC AMB/VRC a, b, c

Suvival (%)

60 40 20 0

Results Survival The survival rates of groups treated with amphotericin B (1.5 mg/kg/day) alone (14 1 days) and amphotericin B (1.5 mg/kg/day) combined with voriconazole (40.0 mg/kg/day) (20 3 days) were signicantly higher (P \ 0.05) than those of the groups untreated (11 days) and treated with voriconazole (40.0 mg/kg/day) alone (11 days). The survival rate of the group treated with amphotericin B combined with voriconazole was signicantly higher (P \ 0.05) than that of the group treated with amphotericin B alone (Fig. 1).

10

20

30

Days after infection

Fig. 1 Cumulative mortality of mice treated at 15 days with amphotericin B AMB (1.5 mg/kg/day), voriconazole VRC (40.0 mg/kg/day), and AMB/VRC, amphotericin B (1.5 mg/ kg/day) combinated with voriconazole (40 mg/kg/day). Mean survival times were estimated by the KaplanMeier method and compared among groups by using the log-rank test. Differences were considered signicant when P \ 0.05. aP \ 0.05 versus control; bP \ 0.05 versus AMB (1.5 mg/kg/day); cP \ 0.05 versus VRC (40.0 mg/kg/day)
5.0

lung

10 )-CFU

4.5

a,b a c

Isolation of C. neoformans in the Lung Tissue There was no signicant reduction (P \ 0.05) in the isolation of C. neoformans in lung tissue in the group treated only with amphotericin B (1.5 mg/kg/day), (4.5 0.7), compared to the untreated group (4.3 0.5). Reduction in the isolation of yeast was signicant (P \ 0.05) in the groups treated only with voriconazole (40.0 mg/kg/day) (3.6 0.4) and amp hotericin B combined with voriconazole (40.0 mg/kg/ day) (3.6 0.3), compared with the untreated group and the group treated only amphotericin B (1.5 mg/kg/ day) (Fig. 2).

Mean (Log

4.0

c,d

3.5

3.0 Untreated AMB VRC AMB/VRC

Treatments
Fig. 2 Isolation of yeast in the lung tissue in mice with Severe Combined Immunodeciency (SCID) inoculated intravenously with 3.0 9 105 viable cells of C. neoformans (ATCC 90112-serotype A). Evaluated after treatments by 15 days with AMB, (amphotericin B 1.5 mg/kg/ day); VRC, (voriconazole 40.0 mg/kg/day); AMB/VRC, (amphotericin B 1.5 mg/kg/ day) combined with (voriconazole 40.0 mg/kg/day). Different letters indicate statistical signicant difference for P \ 0.05. Mann Whitney test

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uconazole, has driven the most appropriate choice of treatments [16]. This study has evaluated the use of amphotericin B and voriconazole alone, and in combination, to treat pulmonary infections caused by C. neoformans in murine models with SCID. Voriconazole, administered alone, did not prolong the survival of the animals, but when administered in combination with amphotericin B, it signicantly increased survival, of animals compared with the untreated group and the group treated only with amphotericin B. In other studies using murine models, it has been demonstrated that the treatment for cryptococcosis with voriconazole (60.0 mg/kg/day) over the course of 15 days effectively increases the survival of the mice treated [15, 17]. Voriconazole administered in combination with amphotericin B has been shown to prolong the survival of mice with experimental cryptococcosis with Severe Combined Immunodeciency (SCID) [18]. Quantication of yeast in the brain is an important marker that may be utilized to verify the fungicide activity of drugs in patients with cryptococcosis of the CNS [19]. There was no signicant reduction in the isolation of C. neoformans in the lung tissue of animals in the group treated with amphotericin B alone, compared to the group untreated. Another study has also shown that amphotericin, administered alone in cases of experimental cryptococcosis, reduce the isolation of C. neoformans in brain tissue [20]. There was a significant reduction in the isolation of yeast in the lung tissue of animals treated with voriconazole alone and amphotericin B combined with voriconazole. Our results are consistent with those reported by Serena et al. [17], who have found that the reduction of C. neoformans in lung and brain tissue is signicant in animals treated with voriconazole at low doses (10 and 40.0 mg/kg/day). Although it was observed that there was no signicant difference between the reduction of yeast in the lung tissue of the group treated only with voriconazole and the lung tissue of the group treated with a combination of amphotericin B and voriconazole, treatment with voriconazole alone did not effectively increase survival, because the survival of animals in this group was similar to survival in the untreated group. Although we nd that none of the treatments eradicated C. neoformans in lung tissue, we suggest that treatment with a combination of amphotericin B (1.5 mg/kg/day) and voriconazole (40.0 mg/kg/day)

may be a better alternative for the treatment for lung infections caused by C. neoformans, under the conditions evaluated in this study. Future studies should be conducted, to determine optimal treatment length and better concentrations of the drugs associated with disease resolution.
Acknowledgments The authors are grateful to Pzer for providing pure voriconazole; to Mr. Carlos Augusto da Silva, the Animal Centers Technician, for his valuable assistance and FAPESP and CNPq, for nancial support. Conict of Interest This study presents no conict of interest.

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