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Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense
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ARTICLE IN PRESS International Journal of Antimicrobial Agents xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense Fabiola Fernández-Silva a , Javier Capilla b , Emilio Mayayo a , Deanna Sutton c , Josep Guarro b,∗ a
Unitat d’Anatomia Patològica, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain c Fungus Testing Laboratory, University of Texas Health Science Center, San Antonio, TX, USA b
a r t i c l e
i n f o
Article history: Received 30 September 2013 Accepted 20 March 2014 Keywords: Antifungal combinations Fungal infection Sarocladium kiliense Animal model
a b s t r a c t Since human infections by Sarocladium (Acremonium) kiliense are usually refractory to antifungal therapy, combinations of drugs are an option that needs exploring. The antifungal activities of anidulafungin plus posaconazole or voriconazole and of amphotericin B plus voriconazole were evaluated and their efficacy was tested in a murine model of disseminated infection by three S. kiliense strains. The results showed that although efficacy was strain-dependent, these combinations work better, in general, than monotherapies. The best results were obtained with the combination of anidulafungin plus posaconazole, although the in vitro assay showed indifference or an antagonistic effect. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Introduction Sarocladium kiliense, formerly known as Acremonium kiliense [1], is a saprobic fungus found worldwide. It is involved in human mycetoma, keratitis and onychomycosis. However, disseminated infections have been reported in immunosuppressed and occasionally in immunocompetent patients. These infections are difficult to treat and often have a fatal outcome [2,3]. The genus comprises around 150 species, with S. kiliense being the most common in clinical samples. This species shows a high minimum inhibitory concentration (MIC) to almost all antifungal drugs [3], which has hindered the optimisation of antifungal treatments. Amphotericin B (AMB) and, to a lesser degree, voriconazole (VRC) are the most commonly used drugs to treat Acremonium infections [4], but therapeutic failure has been reported mainly in cases of fungaemia or disseminated human infection [3,5–7]. Regarding resistance of this fungus and the cases of therapeutic failure reported, other approaches should be considered. Studies performed in an animal model of systemic infection by this fungus have shown posaconazole (PSC) and to a lesser extent anidulafungin (AFG) to have some beneficial effect in increasing survival, with 40% and 10% of animals surviving the
∗ Corresponding author. Present address: Unitat de Microbiologia, Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Carrer Sant Llorenc¸ 21, 43201 Reus, Spain. Tel.: +34 977 759 359; fax: +34 977 759 322. E-mail address: [email protected] (J. Guarro).
infection after PSC and AFG administration, respectively, and both reducing fungal loads from targeted organs in a moderate fashion [8]. Combined therapies have not been evaluated in animals or humans but they could be an alternative worth exploring, particularly for immunosuppressed patients with disseminated infection. Therefore, the aim of this study was to evaluate the in vivo efficacy of combined antifungal agents. Combinations of drugs were selected based on previous experimental monotherapies that had shown modest efficacy, i.e. PSC and AFG [8], and AMB and VRC, which are the drugs most used in the clinical setting. Materials and methods Three clinical strains of S. kiliense (UTHSC 01-2238, UTHSC 033197 and UTHSC 07-550) from the Fungus Testing Laboratory of the University of Texas Health Science Center (San Antonio, TX) were tested. The strains were chosen based on previous results demonstrating low or no response to experimental antifungal monotherapy [8]. Inocula were prepared as previously described [8]. The in vitro susceptibilities of the isolates to AFG, AMB, VRC and PSC were determined by the microdilution reference method for filamentous fungi as described by the Clinical and Laboratory Standards Institute (CLSI) [9]. Drug interactions were assessed using the checkerboard method, and the fractional inhibitory concentration index (FICI) was used to classify drug interactions [10,11]. Four-week-old OF-1 male mice (Charles River, Criffa S.A., Barcelona, Spain) weighing 28–30 g were used. Mice were housed
http://dx.doi.org/10.1016/j.ijantimicag.2014.03.010 0924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Please cite this article in press as: Fernández-Silva F, et al. Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.010
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Fig. 1. Cumulative mortality of mice infected with 2 × 108 conidia/animal of three strains of Sarocladium kiliense. a P < 0.05 vs. control; b P < 0.05 vs. AMB; c P < 0.05 vs. VRC; d P < 0.05 vs. PSC. AFG, anidulafungin; AMB, amphotericin B; PSC, posaconazole; VRC, voriconazole.
in standard boxes with corncob bedding and access to food and water ad libitum. All animal care procedures were supervised and approved by the Animal Welfare and Ethics Committee of Universitat Rovira i Virgili (Reus, Spain). Mice were immunosuppressed 1 day before the infection by intraperitoneal (i.p.) administration of a single dose of 200 mg/kg cyclophosphamide plus a single intravenous (i.v.) dose of 150 mg/kg 5-fluorouracil [12]. The efficacies of the different treatments were evaluated through prolongation of survival and reduction of fungal tissue burden in the spleen and kidneys [13]. Groups of eight mice were established for the different studies. For the survival study, animals were challenged with 2 × 108 conidia in 0.2 mL of 0.9% saline injected into the lateral tail vein and were checked daily for 30 days [8]. Survivors and those mice meeting the criteria for discomfort were euthanised by CO2 inhalation. For tissue burden determination, mice were infected with 2 × 106 conidia in 0.2 mL of 0.9% saline. This inoculum was chosen to avoid the fast killing obtained with the highest inoculum and to allow the fungal load of treated groups to be compared with controls on the same day [8]. The different groups were treated as follows: AMB deoxycholate at 0.8 mg/kg/day i.v. once daily [14]; PSC at 20 mg/kg orally (p.o.) twice a day by gavage [15]; VRC at 40 mg/kg p.o. once daily [16]; or AFG at 10 mg/kg i.p. once daily [17]; AFG + PSC; AFG + VRC; and VRC + AMB. The doses and routes of administration used in the combined therapies were the same as in the monotherapies. From 3 days before the infection, mice receiving VRC were given grapefruit juice instead of water [18]. All treatments were started 24 h after the challenge and lasted for 7 days. Animals were checked for survival twice daily for 30 days. Fungal load was determined after 5 days of treatment to compare the results with the control group since control mice start to die on that day [8]. Animals were euthanised and the kidneys and spleen were removed aseptically [13]. Approximately one-half of each organ was weighed, mechanically homogenised in 1 mL of sterile saline and serially 10-fold diluted. Dilutions were placed on potato dextrose agar medium (Difco, Detroit, IL) and were incubated at 25 ◦ C for 7 days to determine the CFU/g. Mean survival time was estimated using the Kaplan–Meier method and was compared among groups by the log-rank test. Organ burden data were analysed by the Mann–Whitney U-test using GraphPad Prism 4 for Windows (GraphPad Software Inc., La Jolla, CA). P-values of ≤0.05 were considered statistically significant.
Results Table 1 shows the in vitro activity of the different drug combinations assayed and their interactions against the three strains of S. kiliense. In general, the FICI showed an indifferent effect for all of the combinations and strains evaluated, with the exception of strain UTHSC 03-3197 against which the combination AFG + PSC showed antagonism and strain UTHSC 01-2338 against which AFG + VRC showed synergism.
Table 1 In vitro antifungal activity of antifungal drugs and their interactions against three clinical isolates of Sarocladium (Acremonium) kiliense. Antifungal drug
AMB AFG PSC VRC AFG + PSC AFG + VRC AMB + VRC
MIC or MEC (g/mL) [FICI]a UTHSC 01-2238
UTHSC 03-3197
UTHSC 07-550
8 8 16 2 8/8 [2] 1/0.5 [0.37] 8/8 [2]
4 4 16 0.5 8/8 [5] 2/0.25 [1.25] 4/4 [2]
2 16 16 2 8/8 [2] 4/0.25 [0.75] 4/4 [2]
MIC, minimum inhibitory concentration; MEC, minimal effective concentration (AFG only); FICI, fractional inhibitory concentration index; AMB, amphotericin B; AFG, anidulafungin; PSC, posaconazole; VRC, voriconazole. a FICI ≤ 0.5, synergistic; >0.5 to <4, indifferent; and ≥4, antagonistic.
Results of the survival studies are shown in Fig. 1. In general, almost all of the treatments prolonged survival in comparison with the control group, with the exception of VRC and AMB monotherapies. The combinations AFG + PSC and AFG + VRC improved survival of their respective monotherapies for all of the strains tested, with the exception of strain UTHSC 03-3197 for which the AFG + PSC combination was equally efficient as the respective monotherapies. Results of the tissue burden study are shown in Fig. 2. The combined therapy AFG + PSC reduced the fungal load in the kidneys and spleen with respect to the control, the respective monotherapies and the other combinations for two strains (UTHSC 03-3197 and UTHSC 07-550), whilst for strain UTHSC 01-2238 the combination did not improve the results over the monotherapies, with PSC showing the highest efficacy. Discussion The main reasons for the absence of appropriate antifungal therapy for S. kiliense infections are the lack of reliable sequences for identifying the species involved in such infections [19], the few clinical cases that have been reported [3,20] and the few antifungal susceptibility studies carried out so far [2,3,19,20]. Recently, S. kiliense has been reported as the most common species of Sarocladium in clinical samples [19]. Recent experimental studies have determined the virulence [13] and efficacy of different antifungal therapies against isolates of several Acremonium or Sarocladium spp. of clinical interest [8]. These studies showed that S. kiliense is the most virulent species and that PSC, despite having modest efficacy, is better than VRC, AFG and AMB. In the present study, PSC in general showed some efficacy, except in one strain against which PSC was more effective even than the combined treatments. The relatively good results shown by PSC in the present study do not correlate with the high MIC of this drug observed in the in vitro study. The results do, however, corroborate the poor efficacy of the two drugs that are most commonly used currently in the treatment of disseminated infection by S. kiliense, i.e. AMB and VRC. Having
Please cite this article in press as: Fernández-Silva F, et al. Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.010
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Fig. 2. Effects of the antifungal treatments on CFU counts in the spleen and kidneys of mice infected with 2 × 106 conidia/animal of three strains of Sarocladium kiliense: amphotericin B (AMB) at 0.8 mg/kg; posaconazole (PSC) at 20 mg/kg twice a day; voriconazole (VRC) at 40 mg/kg; anidulafungin (AFG) at 10 mg/kg. a P < 0.05 vs. control; b P < 0.05 vs. AFG; c P < 0.05 vs. AMB; d P < 0.05 vs. VRC; e P < 0.05 vs. PSC; f P < 0.05 vs. AFG + PSC; g P < 0.05 vs. AFG + VRC; h P < 0.05 vs. AMB + VRC. Horizontal lines indicate mean values.
said that, AMB has only been tested in its deoxycholate formulation and there are no data on its lipid formulation, which might indeed improve on the low efficacy shown by the traditional formulation. To our knowledge, this is the first study that has explored the efficacy of different antifungal combinations in the treatment of S. kiliense infections. Although only a small number of strains were tested, this study shows the poor predictive value of the in vitro data of drug combinations. The interactions of the drugs were mainly indifferent, and only against one strain (UTHSC 01-2238)
did the combination AFG + VRC show in vitro synergism. A synergistic effect for this combination has been reported previously in vitro [21] as well as in vivo [17] for Aspergillus infections. However, the combination AFG + VRC was not able to improve efficacy over their respective monotherapies in the animal model. By contrast, the combination AFG + PSC, although being indifferent or antagonistic in vitro, showed the highest efficacy in two of the three experimental infections, also confirming the poor correlation between in vitro and in vivo data. Although mice given grapefruit juice have been
Please cite this article in press as: Fernández-Silva F, et al. Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.010
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shown to be a valid model for the assay of VRC efficacy, this might in fact be considered a limitation of the study. Data would need to be carefully interpreted because it has been suggested that grapefruit juice might cause inhibition of fungal cytochrome enzymes or possibly stimulation of the protective host immune response, although this has not yet been demonstrated experimentally [18]. Overall, the promising results obtained with AFG + PSC in improving survival and reducing the fungal load deserve further studies to evaluate any potential role of this combination for the treatment of this refractory infection. Funding: This work was partially funded by the Universitat Rovira i Virgili (Reus, Spain). Competing interests: None declared. Ethical approval: Procedures were supervised and approved by Luis Loriente Sanz (ID 39671243) of the Veterinary and Animal Welfare Advisory of the Universitat Rovira i Virgili Animal Welfare and Ethics Committee (Reus, Spain). References [1] Summerbell RC, Gueidan C, Schroers HJ, de Hoog GS, Starink M, Rosete YA, et al. Acremonium phylogenetic overview and revision of Gliomastix, Sarocladium, and Trichothecium. Stud Mycol 2011;68:139–62. [2] Guarro J, Gams W, Pujol I, Gené J. Acremonium species: new emerging fungal opportunists—in vitro antifungal susceptibilities and review. Clin Infect Dis 1997;25:1222–9. [3] Khan Z, Al-Obaid K, Ahmad S, Ghani AA, Joseph L, Chandy R. Acremonium kiliense: reappraisal of its clinical significance. J Clin Microbiol 2011;49:2342–7. [4] Mattei D, Mordini N, Lo Nigro C, Gallamini A, Osenda M, Pugno F, et al. Successful treatment of Acremonium fungemia with voriconazole. Mycoses 2003;46:511–14. [5] Beaudreuil S, Buchler M, Al Najjar A, Bastides F, Francois M, Duong TH, et al. Acute septic arthritis after kidney transplantation due to Acremonium. Nephrol Dial Transplant 2003;18:850–1. [6] Guarro J, Del Palacio A, Gené J, Cano J, González CG. A case of colonization of a prosthetic mitral valve by Acremonium strictum. Rev Iberoam Micol 2009;26:146–8. [7] Hitoto H, Pihet M, Weil B, Chabasse D, Bouchara JP, Rachieru-Sourisseau P. Acremonium strictum fungaemia in a paediatric immunocompromised patient: diagnosis and treatment difficulties. Mycopathologia 2010;170:161–4.
[8] Fernández-Silva F, Capilla J, Mayayo E, Sutton DA, Hernández P, Guarro J. Evaluation of the efficacies of amphotericin B, posaconazole, voriconazole, and anidulafungin in a murine disseminated infection by the emerging opportunistic fungus Sarocladium (Acremonium) kiliense. Antimicrob Agents Chemother 2013;57:6265–9. [9] Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. Document M38-A2. 2nd ed. Wayne, PA: CLSI; 2008. [10] Dannaoui E, Lortholary O, Dromer F. In vitro evaluation of double and triple combinations of antifungal drugs against Aspergillus fumigatus and Aspergillus terreus. Antimicrob Agents Chemother 2004;48:970–8. [11] Johnson MD, MacDougall C, Ostrosky-Zeichner L, Perfect JR, Rex JH. Combination antifungal therapy. Antimicrob Agents Chemother 2004;48: 693–715. [12] Ortoneda M, Capilla J, Pastor FJ, Serena C, Guarro J. Interaction of granulocyte colony-stimulating factor and high doses of liposomal amphotericin B in the treatment of systemic murine scedosporiosis. Diagn Microbiol Infect Dis 2004;50:247–51. [13] Fernández-Silva F, Capilla J, Mayayo E, Sutton DA, Guarro J. Experimental murine acremoniosis: an emerging opportunistic human infection. Med Mycol 2014;52:29–35. [14] Graybill JR. The role of murine models in the development of antifungal therapy for systemic mycoses. Drug Resist Updat 2000;3:364–83. [15] Rodríguez MM, Pastor FJ, Calvo E, Salas V, Sutton DA, Guarro J. Correlation of in vitro activity, serum levels, and in vivo efficacy of posaconazole against Rhizopus microsporus in a murine disseminated infection. Antimicrob Agents Chemother 2009;53:5022–5. [16] Rodríguez MM, Pastor FJ, Salas V, Calvo E, Mayayo E, Guarro J. Experimental murine scedosporiosis: histopathology and azole treatment. Antimicrob Agents Chemother 2010;54:3980–4. [17] Calvo E, Pastor FJ, Mayayo E, Salas V, Guarro J. In vitro activity and in vivo efficacy of anidulafungin in murine infections by Aspergillus flavus. Antimicrob Agents Chemother 2011;55:1290–2. [18] Graybill JR, Najvar LK, Gonzalez GM, Hernandez S, Bocanegra R. Improving the mouse model for studying the efficacy of voriconazole. J Antimicrob Chemother 2003;51:1373–6. [19] Perdomo H, Sutton DA, García D, Fothergill AW, Cano J, Gené J, et al. Spectrum of clinically relevant Acremonium species in the United States. J Clin Microbiol 2011;49:243–56. [20] Das S, Saha R, Dar SA, Ramachandran VG. Acremonium species: a review of the etiological agents of emerging hyalohyphomycosis. Mycopathologia 2010;170:361–75. [21] Perkhofer S, Jost D, Dierich MP, Lass-Flörl C. Susceptibility testing of anidulafungin and voriconazole alone and in combination against conidia and hyphae of Aspergillus spp. under hypoxic conditions. Antimicrob Agents Chemother 2008;52:1873–5.
Please cite this article in press as: Fernández-Silva F, et al. Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.010
ARTICLE IN PRESS International Journal of Antimicrobial Agents xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense Fabiola Fernández-Silva a , Javier Capilla b , Emilio Mayayo a , Deanna Sutton c , Josep Guarro b,∗ a
Unitat d’Anatomia Patològica, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain c Fungus Testing Laboratory, University of Texas Health Science Center, San Antonio, TX, USA b
a r t i c l e
i n f o
Article history: Received 30 September 2013 Accepted 20 March 2014 Keywords: Antifungal combinations Fungal infection Sarocladium kiliense Animal model
a b s t r a c t Since human infections by Sarocladium (Acremonium) kiliense are usually refractory to antifungal therapy, combinations of drugs are an option that needs exploring. The antifungal activities of anidulafungin plus posaconazole or voriconazole and of amphotericin B plus voriconazole were evaluated and their efficacy was tested in a murine model of disseminated infection by three S. kiliense strains. The results showed that although efficacy was strain-dependent, these combinations work better, in general, than monotherapies. The best results were obtained with the combination of anidulafungin plus posaconazole, although the in vitro assay showed indifference or an antagonistic effect. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Introduction Sarocladium kiliense, formerly known as Acremonium kiliense [1], is a saprobic fungus found worldwide. It is involved in human mycetoma, keratitis and onychomycosis. However, disseminated infections have been reported in immunosuppressed and occasionally in immunocompetent patients. These infections are difficult to treat and often have a fatal outcome [2,3]. The genus comprises around 150 species, with S. kiliense being the most common in clinical samples. This species shows a high minimum inhibitory concentration (MIC) to almost all antifungal drugs [3], which has hindered the optimisation of antifungal treatments. Amphotericin B (AMB) and, to a lesser degree, voriconazole (VRC) are the most commonly used drugs to treat Acremonium infections [4], but therapeutic failure has been reported mainly in cases of fungaemia or disseminated human infection [3,5–7]. Regarding resistance of this fungus and the cases of therapeutic failure reported, other approaches should be considered. Studies performed in an animal model of systemic infection by this fungus have shown posaconazole (PSC) and to a lesser extent anidulafungin (AFG) to have some beneficial effect in increasing survival, with 40% and 10% of animals surviving the
∗ Corresponding author. Present address: Unitat de Microbiologia, Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Carrer Sant Llorenc¸ 21, 43201 Reus, Spain. Tel.: +34 977 759 359; fax: +34 977 759 322. E-mail address: [email protected] (J. Guarro).
infection after PSC and AFG administration, respectively, and both reducing fungal loads from targeted organs in a moderate fashion [8]. Combined therapies have not been evaluated in animals or humans but they could be an alternative worth exploring, particularly for immunosuppressed patients with disseminated infection. Therefore, the aim of this study was to evaluate the in vivo efficacy of combined antifungal agents. Combinations of drugs were selected based on previous experimental monotherapies that had shown modest efficacy, i.e. PSC and AFG [8], and AMB and VRC, which are the drugs most used in the clinical setting. Materials and methods Three clinical strains of S. kiliense (UTHSC 01-2238, UTHSC 033197 and UTHSC 07-550) from the Fungus Testing Laboratory of the University of Texas Health Science Center (San Antonio, TX) were tested. The strains were chosen based on previous results demonstrating low or no response to experimental antifungal monotherapy [8]. Inocula were prepared as previously described [8]. The in vitro susceptibilities of the isolates to AFG, AMB, VRC and PSC were determined by the microdilution reference method for filamentous fungi as described by the Clinical and Laboratory Standards Institute (CLSI) [9]. Drug interactions were assessed using the checkerboard method, and the fractional inhibitory concentration index (FICI) was used to classify drug interactions [10,11]. Four-week-old OF-1 male mice (Charles River, Criffa S.A., Barcelona, Spain) weighing 28–30 g were used. Mice were housed
http://dx.doi.org/10.1016/j.ijantimicag.2014.03.010 0924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Please cite this article in press as: Fernández-Silva F, et al. Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.010
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Fig. 1. Cumulative mortality of mice infected with 2 × 108 conidia/animal of three strains of Sarocladium kiliense. a P < 0.05 vs. control; b P < 0.05 vs. AMB; c P < 0.05 vs. VRC; d P < 0.05 vs. PSC. AFG, anidulafungin; AMB, amphotericin B; PSC, posaconazole; VRC, voriconazole.
in standard boxes with corncob bedding and access to food and water ad libitum. All animal care procedures were supervised and approved by the Animal Welfare and Ethics Committee of Universitat Rovira i Virgili (Reus, Spain). Mice were immunosuppressed 1 day before the infection by intraperitoneal (i.p.) administration of a single dose of 200 mg/kg cyclophosphamide plus a single intravenous (i.v.) dose of 150 mg/kg 5-fluorouracil [12]. The efficacies of the different treatments were evaluated through prolongation of survival and reduction of fungal tissue burden in the spleen and kidneys [13]. Groups of eight mice were established for the different studies. For the survival study, animals were challenged with 2 × 108 conidia in 0.2 mL of 0.9% saline injected into the lateral tail vein and were checked daily for 30 days [8]. Survivors and those mice meeting the criteria for discomfort were euthanised by CO2 inhalation. For tissue burden determination, mice were infected with 2 × 106 conidia in 0.2 mL of 0.9% saline. This inoculum was chosen to avoid the fast killing obtained with the highest inoculum and to allow the fungal load of treated groups to be compared with controls on the same day [8]. The different groups were treated as follows: AMB deoxycholate at 0.8 mg/kg/day i.v. once daily [14]; PSC at 20 mg/kg orally (p.o.) twice a day by gavage [15]; VRC at 40 mg/kg p.o. once daily [16]; or AFG at 10 mg/kg i.p. once daily [17]; AFG + PSC; AFG + VRC; and VRC + AMB. The doses and routes of administration used in the combined therapies were the same as in the monotherapies. From 3 days before the infection, mice receiving VRC were given grapefruit juice instead of water [18]. All treatments were started 24 h after the challenge and lasted for 7 days. Animals were checked for survival twice daily for 30 days. Fungal load was determined after 5 days of treatment to compare the results with the control group since control mice start to die on that day [8]. Animals were euthanised and the kidneys and spleen were removed aseptically [13]. Approximately one-half of each organ was weighed, mechanically homogenised in 1 mL of sterile saline and serially 10-fold diluted. Dilutions were placed on potato dextrose agar medium (Difco, Detroit, IL) and were incubated at 25 ◦ C for 7 days to determine the CFU/g. Mean survival time was estimated using the Kaplan–Meier method and was compared among groups by the log-rank test. Organ burden data were analysed by the Mann–Whitney U-test using GraphPad Prism 4 for Windows (GraphPad Software Inc., La Jolla, CA). P-values of ≤0.05 were considered statistically significant.
Results Table 1 shows the in vitro activity of the different drug combinations assayed and their interactions against the three strains of S. kiliense. In general, the FICI showed an indifferent effect for all of the combinations and strains evaluated, with the exception of strain UTHSC 03-3197 against which the combination AFG + PSC showed antagonism and strain UTHSC 01-2338 against which AFG + VRC showed synergism.
Table 1 In vitro antifungal activity of antifungal drugs and their interactions against three clinical isolates of Sarocladium (Acremonium) kiliense. Antifungal drug
AMB AFG PSC VRC AFG + PSC AFG + VRC AMB + VRC
MIC or MEC (g/mL) [FICI]a UTHSC 01-2238
UTHSC 03-3197
UTHSC 07-550
8 8 16 2 8/8 [2] 1/0.5 [0.37] 8/8 [2]
4 4 16 0.5 8/8 [5] 2/0.25 [1.25] 4/4 [2]
2 16 16 2 8/8 [2] 4/0.25 [0.75] 4/4 [2]
MIC, minimum inhibitory concentration; MEC, minimal effective concentration (AFG only); FICI, fractional inhibitory concentration index; AMB, amphotericin B; AFG, anidulafungin; PSC, posaconazole; VRC, voriconazole. a FICI ≤ 0.5, synergistic; >0.5 to <4, indifferent; and ≥4, antagonistic.
Results of the survival studies are shown in Fig. 1. In general, almost all of the treatments prolonged survival in comparison with the control group, with the exception of VRC and AMB monotherapies. The combinations AFG + PSC and AFG + VRC improved survival of their respective monotherapies for all of the strains tested, with the exception of strain UTHSC 03-3197 for which the AFG + PSC combination was equally efficient as the respective monotherapies. Results of the tissue burden study are shown in Fig. 2. The combined therapy AFG + PSC reduced the fungal load in the kidneys and spleen with respect to the control, the respective monotherapies and the other combinations for two strains (UTHSC 03-3197 and UTHSC 07-550), whilst for strain UTHSC 01-2238 the combination did not improve the results over the monotherapies, with PSC showing the highest efficacy. Discussion The main reasons for the absence of appropriate antifungal therapy for S. kiliense infections are the lack of reliable sequences for identifying the species involved in such infections [19], the few clinical cases that have been reported [3,20] and the few antifungal susceptibility studies carried out so far [2,3,19,20]. Recently, S. kiliense has been reported as the most common species of Sarocladium in clinical samples [19]. Recent experimental studies have determined the virulence [13] and efficacy of different antifungal therapies against isolates of several Acremonium or Sarocladium spp. of clinical interest [8]. These studies showed that S. kiliense is the most virulent species and that PSC, despite having modest efficacy, is better than VRC, AFG and AMB. In the present study, PSC in general showed some efficacy, except in one strain against which PSC was more effective even than the combined treatments. The relatively good results shown by PSC in the present study do not correlate with the high MIC of this drug observed in the in vitro study. The results do, however, corroborate the poor efficacy of the two drugs that are most commonly used currently in the treatment of disseminated infection by S. kiliense, i.e. AMB and VRC. Having
Please cite this article in press as: Fernández-Silva F, et al. Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.010
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Fig. 2. Effects of the antifungal treatments on CFU counts in the spleen and kidneys of mice infected with 2 × 106 conidia/animal of three strains of Sarocladium kiliense: amphotericin B (AMB) at 0.8 mg/kg; posaconazole (PSC) at 20 mg/kg twice a day; voriconazole (VRC) at 40 mg/kg; anidulafungin (AFG) at 10 mg/kg. a P < 0.05 vs. control; b P < 0.05 vs. AFG; c P < 0.05 vs. AMB; d P < 0.05 vs. VRC; e P < 0.05 vs. PSC; f P < 0.05 vs. AFG + PSC; g P < 0.05 vs. AFG + VRC; h P < 0.05 vs. AMB + VRC. Horizontal lines indicate mean values.
said that, AMB has only been tested in its deoxycholate formulation and there are no data on its lipid formulation, which might indeed improve on the low efficacy shown by the traditional formulation. To our knowledge, this is the first study that has explored the efficacy of different antifungal combinations in the treatment of S. kiliense infections. Although only a small number of strains were tested, this study shows the poor predictive value of the in vitro data of drug combinations. The interactions of the drugs were mainly indifferent, and only against one strain (UTHSC 01-2238)
did the combination AFG + VRC show in vitro synergism. A synergistic effect for this combination has been reported previously in vitro [21] as well as in vivo [17] for Aspergillus infections. However, the combination AFG + VRC was not able to improve efficacy over their respective monotherapies in the animal model. By contrast, the combination AFG + PSC, although being indifferent or antagonistic in vitro, showed the highest efficacy in two of the three experimental infections, also confirming the poor correlation between in vitro and in vivo data. Although mice given grapefruit juice have been
Please cite this article in press as: Fernández-Silva F, et al. Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.010
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shown to be a valid model for the assay of VRC efficacy, this might in fact be considered a limitation of the study. Data would need to be carefully interpreted because it has been suggested that grapefruit juice might cause inhibition of fungal cytochrome enzymes or possibly stimulation of the protective host immune response, although this has not yet been demonstrated experimentally [18]. Overall, the promising results obtained with AFG + PSC in improving survival and reducing the fungal load deserve further studies to evaluate any potential role of this combination for the treatment of this refractory infection. Funding: This work was partially funded by the Universitat Rovira i Virgili (Reus, Spain). Competing interests: None declared. Ethical approval: Procedures were supervised and approved by Luis Loriente Sanz (ID 39671243) of the Veterinary and Animal Welfare Advisory of the Universitat Rovira i Virgili Animal Welfare and Ethics Committee (Reus, Spain). References [1] Summerbell RC, Gueidan C, Schroers HJ, de Hoog GS, Starink M, Rosete YA, et al. Acremonium phylogenetic overview and revision of Gliomastix, Sarocladium, and Trichothecium. Stud Mycol 2011;68:139–62. [2] Guarro J, Gams W, Pujol I, Gené J. Acremonium species: new emerging fungal opportunists—in vitro antifungal susceptibilities and review. Clin Infect Dis 1997;25:1222–9. [3] Khan Z, Al-Obaid K, Ahmad S, Ghani AA, Joseph L, Chandy R. Acremonium kiliense: reappraisal of its clinical significance. J Clin Microbiol 2011;49:2342–7. [4] Mattei D, Mordini N, Lo Nigro C, Gallamini A, Osenda M, Pugno F, et al. Successful treatment of Acremonium fungemia with voriconazole. Mycoses 2003;46:511–14. [5] Beaudreuil S, Buchler M, Al Najjar A, Bastides F, Francois M, Duong TH, et al. Acute septic arthritis after kidney transplantation due to Acremonium. Nephrol Dial Transplant 2003;18:850–1. [6] Guarro J, Del Palacio A, Gené J, Cano J, González CG. A case of colonization of a prosthetic mitral valve by Acremonium strictum. Rev Iberoam Micol 2009;26:146–8. [7] Hitoto H, Pihet M, Weil B, Chabasse D, Bouchara JP, Rachieru-Sourisseau P. Acremonium strictum fungaemia in a paediatric immunocompromised patient: diagnosis and treatment difficulties. Mycopathologia 2010;170:161–4.
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Please cite this article in press as: Fernández-Silva F, et al. Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense. Int J Antimicrob Agents (2014), http://dx.doi.org/10.1016/j.ijantimicag.2014.03.010