JAC Advance Access published online on May 20, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn215
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Original research |
Combined therapy in treatment of murine infection by Fusarium solani
Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Spain
* Correspondence address. Unitat de Microbiologia, Facultat de Medicina, Universitat Rovira i Virgili, Carrer Sant Llorenç, 21, 43201 Reus, Spain. Tel: +34-977-759359; Fax: +34-977-759322; E-mail: josep.guarro{at}urv.cat
Received 4 February 2008; returned 14 March 2008; revised 18 April 2008; accepted 25 April 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Objectives: We evaluated the efficacy of voriconazole, amphotericin B and micafungin alone and combined in a murine model of disseminated infection by Fusarium solani.
Methods: Immunosuppressed mice were treated with voriconazole at 60 mg/kg/day, amphotericin B at 3 mg/kg/day, micafungin at 10 mg/kg/day or combinations of these antifungal drugs. For survival studies, treatment began 1 day after infection and continued for 10 days. For tissue burden studies, animals were sacrificed on day 6 of the treatment and the fungal load in the kidneys and spleens was measured. The experiments were carried out using two different clinical strains of F. solani.
Results: Only the combination of voriconazole plus amphotericin B prolonged the survival of the mice versus the controls for the two strains tested. However, this combination only reduced tissue burden in the kidney and spleen of mice infected by one strain. The other treatments were clearly less effective.
Conclusions: Voriconazole plus amphotericin B may have a role in the treatment of fusariosis.
Key Words: voriconazole , amphotericin B , micafungin , murine model
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Several species of Fusarium can cause invasive fungal infections, mainly in patients with underlying haematological malignancies or haematopoietic stem cell transplantation.1–4 These infections are commonly refractory to treatment and show a high mortality rate. Most of the cases of fusariosis are due to Fusarium solani, which seems to be the most virulent species of the genus, at least for mice,5 and also the most resistant to antifungals.1,6 Although F. solani showed universal resistance to antifungal drugs in an in vitro study,7 primary therapies with voriconazole or with lipid formulations of amphotericin B are currently recommended to treat fusariosis.8 Some clinical trials have shown that these drugs are effective, although against which species of Fusarium is unknown. In two studies that treated 11 and 21 patients with voriconazole, the overall response was 43% to 45%.9,10 In another clinical trial, of 11 patients with fusariosis treated with amphotericin B lipid complex, 9 had a complete or partial response.11 On the other hand, treatment with these drugs, especially different formulations of amphotericin B, failed in many other cases of fusariosis.3,11 Interestingly, in two recent cases, the combination of voriconazole and amphotericin B led to clinical improvement before neutropenia resolved.12,13 In order to assess whether this limited success was merely anecdotal or not, since in both cases the species involved were different, we have evaluated the efficacy of this combined therapy. Combinations of each of the two drugs with an echinocandin were also tested.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Fungi
Two clinical isolates, FMR 7995 and FMR 4391, belonging to two different genetic clades of the F. solani species complex7 were used in this study. The isolates were stored at –80°C in potato dextrose broth with glycerol, and prior to testing they were subcultured on potato dextrose agar at 30°C. On the day of infection, 5 day cultures on potato dextrose agar were suspended in sterile saline and filtered through sterile gauze to remove hyphae. The resulting suspensions were adjusted to the desired inoculum size with a haemocytometer. Dilutions of the original suspension were cultured on potato dextrose agar plates to confirm the counts determined with the haemocytometer.
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 corn cob bedding and had free access to food and water. All experiments were performed in accordance with the highest standards for care and treatment of research animals, as approved by the Animal Welfare Committee of the Rovira i Virgili University.
The drugs tested were amphotericin B, purchased as Fungizona (Bristol-Myers Squibb, S.L., Madrid, Spain); micafungin, provided by Astellas Pharma Inc., Tokyo, Japan; and voriconazole, purchased as Vfend (Pfizer S.A., Madrid, Spain).
Mice were immunosuppressed by a single intraperitoneal (ip) injection of 200 mg of cyclophosphamide (Genoxal; Laboratorios Funk S.A., Barcelona, Spain) per kilogram of body weight plus a single intravenous injection of 150 mg of 5-fluorouracil (Fluoro-uracil; Productos Roche S.A., Madrid, Spain) per kilogram on the same day of challenge.14,15
Mice were challenged with 5 x 103 or 6.6 x 104 conidia/mL of the strain FMR 7995 or FMR 4391, respectively, in 0.2 mL of sterile saline, injected into the lateral tail vein. These inocula were chosen on the basis of a preliminary study because they caused the death of 90% of animals 8 or 9 days after the challenge (data not shown).
For survival studies, groups of 10 mice were randomly established for each strain, treatment and as a control group before starting. Treatment began 1 day after infection and continued for 10 days. Amphotericin B was given at 3 mg/kg of body weight intraperitoneally in a volume of 0.2 mL; micafungin at 10 mg/kg of body weight was given subcutaneously in a volume of 0.1 mL; and voriconazole at 60 mg/kg of body weight was given orally by gavage in a volume of 0.2 mL. Mice also received the following drug combinations: amphotericin B/voriconazole, voriconazole/micafungin and amphotericin B/micafungin at the doses described above. Control animals received no treatment. From day 3 prior to infection, the mice that received voriconazole and the control mice were given 100% grapefruit juice (Hero, Spain) instead of water.16 The mice in each experiment were checked daily for 30 days after the challenge.
For tissue burden studies, groups of five mice were randomly established for each strain, treatment and as a control group. All five animals from each group and each treatment were sacrificed by CO2 inhalation on day 6 post-infection. The spleens and kidneys were aseptically removed and homogenized in 1 mL of sterile saline. Serial 10-fold dilutions of the homogenates were plated on potato dextrose agar, incubated for 48 h at 30°C and examined daily for 3 days. The number of cfu per gram of tissue was calculated. This experiment was repeated and the data of the two independent experiments were combined.
Survival rates were evaluated by the Kaplan–Meier method and compared among groups using the log-rank test. Colony counts in tissue burden studies were analysed using the Mann–Whitney U-test. Calculations were made using SPSS 14.0 and GraphPad 4.0 for Windows. A P value of 
0.01 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
The in vitro antifungal activities of amphotericin B, voriconazole and micafungin against the strains FMR 7995 and FMR 4391 of F. solani, determined in a previous study, were 2, 8 and >16 mg/L, and 4, 4 and 64 mg/L, respectively.7 Figure 1 shows the effects of the different treatments on mice survival. For strain FMR 7995, only amphotericin B/voriconazole and amphotericin B significantly prolonged survival versus the control group (P = 0.0004 and P = 0.004). For strain 4391, only treatment with the combination of voriconazole/micafungin (P = 0.0001) and amphotericin B/voriconazole (P = 0.01) significantly prolonged survival versus the control group.
|
Figure 2 shows the effects of the treatments on the fungal load of mice. We tested amphotericin B, voriconazole, micafungin, amphotericin B/voriconazole and voriconazole/micafungin. None of the five treatments tested reduced the tissue burden in any of the organs studied for strain FMR 7995. However, for strain 4391, amphotericin B/voriconazole significantly reduced tissue burden in kidneys and spleen (P = 0.002 and P = 0.006, respectively).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Although the results were not impressive, the present study demonstrated some efficacy of the amphotericin B/voriconazole combination in a murine model of F. solani infection. Despite the high doses of both compounds, the results are only modest: the tissue burden decreased, but minimally so, and only in one strain. In a separate experiment that used animals that were not infected but treated with the same drugs and doses as here, none of them died (data not shown). Several authors have already evaluated the efficacy of each of the two drugs in murine models. Graybill et al.16 demonstrated that voriconazole showed some efficacy in a murine-disseminated infection by a strain of F. solani, although they studied only doses of 10 and 20 mg/kg/day and the degree of protection was modest. The other combination using voriconazole tested in our study, voriconazole plus micafungin, also performed poorly but demonstrated some degree of effectiveness in one strain. Therapy with amphotericin B has been more widely investigated and several animal studies have shown that the efficacy of this drug and its lipid formulations is limited or null.17–19 A few clinical cases have been successfully treated with voriconazole.9,20,21 However, in all the cases that resolved, with one exception,9 the species involved was not F. solani. The combination of voriconazole with amphotericin B has already demonstrated synergy against three species of Fusarium in an in vitro study.22
One of the difficulties of the present study was to develop a suitable model for evaluating the efficacy of the compounds tested. Since Fusarium is not highly pathogenic for immunocompetent individuals, the inocula had to be extremely high. Thus, in previous studies that tested immunocompetent mice, we had to use inocula of 109 conidia/mL for Fusarium verticillioides23 or 107 conidia/mL for F. solani to obtain an acute infection.18 Despite the fact that the animals were immunosuppressed, the fungal load in the various organs was very low in the present study. In the animals that were immunosuppressed 1 day before the challenge, the fungal load in the various organs was very low and not enough to reveal any improvement due to the different treatments (data not shown). Only when the animals were immunosuppressed on the same day as the challenge was the degree of infection considered to be useful for the experiment.
The difficulties in obtaining a suitable experimental murine model of fusariosis have already been indicated by Odds et al.24 Very few of the mice infected with Fusarium oxysporum and immunosuppressed with cyclophosphamide at doses of 150 mg/kg on day –1 died, even with an inoculum of 108 cfu/mL.24
Although we have demonstrated that in vitro antifungal resistance is generalized in the cryptic species of the F. solani complex,7 here we wanted to study in vivo two strains belonging to different genetic clades in order to obtain more conclusive results. The results obtained with both strains were similar and demonstrated that we are still a long way from developing an effective treatment against F. solani infections.
In conclusion, a combination of voriconazole and amphotericin B showed some degree of effectiveness in this murine model. Further studies are needed to assess whether adjusting the doses and the duration of the treatment can improve the modest efficacy of the combination. However, it should be taken into account that in invasive fusariosis, regardless of the treatment used, resolution of myelosuppression is crucial if the infection is to be cured.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
This work was supported by a grant from Fondo de Investigaciones Sanitarias from the Ministerio de Sanidad y Consumo of Spain (PI 050031).
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
None to declare.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
1 . Nucci M, Anaissie E. Fusarium infections in immunocompromised patients. Clin Microbiol Rev (2007) 20:695–704.
2 . Guarro J, Gené J. Opportunistic fusarial infections in humans. Eur J Clin Microbiol Infect Dis (1995) 14:741–54.[CrossRef][Web of Science][Medline]
3
.
Boutati EI, Anaissie EJ. Fusarium, a significant emerging pathogen in patients with hematologic malignancy: ten years experience at a cancer center and implications for management. Blood (1997) 90:999–1008.
4 . Nucci M, Marr KA, Queiroz-Telles F, et al. Fusarium infection in hematopoietic stem cell transplant recipients. Clin Infect Dis (2004) 38:1237–42.[CrossRef][Web of Science][Medline]
5
.
Mayayo E, Pujol I, Guarro J. Experimental pathogenicity of four opportunistic Fusarium species in a murine model. J Med Microbiol (1999) 48:363–6.
6 . Pujol I, Guarro J, Sala J, et al. Effects of incubation temperature, inoculum size, and time of reading on broth microdilution susceptibility test results for amphotericin B against Fusarium. Antimicrob Agents Chemother (1997) 41:808–11.[Abstract]
7
.
Azor M, Gené J, Cano J, et al. Universal in vitro antifungal resistance of genetic clades of the Fusarium solani species complex. Antimicrob Agents Chemother (2007) 51:1500–3.
8 . Pfaller MA, Pappas PG, Wingard JR. Invasive fungal pathogens: current epidemiological trends. Clin Infect Dis (2006) 43:3–14.[CrossRef]
9 . Perfect JR, Marr KA, Walsh TJ, et al. Voriconazole treatment for less-common, emerging or refractory fungal infections. Clin Infect Dis (2003) 36:1122–31.[CrossRef][Web of Science][Medline]
10 . Johnson LB, Kauffman CA. Voriconazole: a new triazole antifungal agent. Clin Infect Dis (2003) 36:630–7.[CrossRef][Web of Science][Medline]
11 . Walsh JT, Hiemenz JW, Seibel NL, et al. Amphotericin B lipid complex for invasive fungal infections: analysis of safety and efficacy in 556 cases. Clin Infect Dis (1998) 26:1383–96.[Web of Science][Medline]
12 . Rodriguez CA, Luján-Zilbermann J, Woodard P, et al. Successful treatment of disseminated fusariosis. Bone Marrow Transplant (2003) 31:411–2.[CrossRef][Web of Science][Medline]
13 . Ho DY, Lee JD, Rosso F, et al. Treating disseminated fusariosis: amphotericin B, voriconazole or both? Mycoses (2007) 50:227–31.[CrossRef][Web of Science][Medline]
14
.
Sun Q, Najvar L, Bocanegra R, et al. In vivo activity of posaconazole against Mucor spp. in an immunosuppressed mouse model. Antimicrob Agents Chemother (2002) 46:2310–2.
15
.
Graybill JR, Bocanegra R, Najvar LK, et al. Granulocyte colony-stimulating factor and azole antifungal therapy in murine aspergillosis: role of immune suppression. Antimicrob Agents Chemother (1998) 42:2467–73.
16
.
Graybill JR, Najvar LK, Gonzalez GM, et al. Improving the mouse model for studying the efficacy of voriconazole. J Antimicrob Chemother (2003) 51:1373–6.
17 . Anaissie EJ, Hachem R, Legrand C, et al. Lack of activity of amphotericin B in systemic murine fusarial infection. J Infect Dis (1992) 165:1155–7.[Web of Science][Medline]
18
.
Guarro J, Pujol I, Mayayo E. In vitro and in vivo experimental activities of antifungal agents against Fusarium solani. Antimicrob Agents Chemother (1999) 43:1256–7.
19
.
Spellberg B, Schwartz J, Fu Y, et al. Comparison of antifungal treatments for murine fusariosis. J Antimicrob Chemother (2006) 58:973–9.
20 . Consigny S, Dhedin N, Datry N, et al. Successful voriconazole treatment of disseminated Fusarium infection in an immunocompromised patient. Clin Infect Dis (2003) 37:311–3.[CrossRef][Web of Science][Medline]
21
.
Sagnelli C, Fumagalli L, Prigitano A, et al. Successful voriconazole therapy of disseminated Fusarium verticillioides infection in an immunocompromised patient receiving chemotherapy. J Antimicrob Chemother (2006) 57:796–8.
22 . Ortoneda M, Capilla J, Pastor FJ, et al. In vitro interactions of licensed and novel antifungal drugs against Fusarium spp. Diagn Microbiol Infect Dis (2004) 48:69–71.[CrossRef][Web of Science][Medline]
23
.
Ortoneda M, Capilla J, Pastor FJ, et al. Efficacy of liposomal amphotericin B in treatment of systemic murine fusariosis. Antimicrob Agents Chemother (2002) 46:2273–5.
24
.
Odds FC, Van Gerven F, Espinel-Ingroff A, et al. Evaluation of possible correlations between antifungal susceptibilities of filamentous fungi in vitro and antifungal treatment outcomes in animal infection models. Antimicrob Agents Chemother (1998) 42:282–8.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Ruiz-Cendoya, M. Marine, M. M. Rodriguez, and J. Guarro Interactions between Triazoles and Amphotericin B in Treatment of Disseminated Murine Infection by Fusarium oxysporum Antimicrob. Agents Chemother., April 1, 2009; 53(4): 1705 - 1708. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||