Journal of Antimicrobial Chemotherapy Advance Access published June 30, 2014
J Antimicrob Chemother doi:10.1093/jac/dku224
Assessing micafungin/triazole combinations for the treatment of invasive scedosporiosis due to Scedosporium apiospermum and Scedosporium boydii Michaela Lackner1*†, Fabiola Ferna´ndez-Silva2†, Josep Guarro3 and Cornelia Lass-Flo¨rl1 1 Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria; 2Unitat d’ Anatomia Patolo`gica, Facultat d’ Medicina i Cie`ncies de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain; 3Unitat d’ Microbiologia, Facultat d’ Medicina i Cie`ncies de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain
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*Corresponding author. Tel: +43-(0)512-9003-70725; Fax: +43-(0)512-9003-73700; E-mail: [email protected] †Both authors contributed equally and share the first author position.
Received 15 April 2014; returned 3 May 2014; revised 13 May 2014; accepted 23 May 2014 Objectives: Scedosporium infections are associated with high therapeutic failure rates. Combination therapy may be an alternative approach to improve outcome. The in vitro and in vivo efficacy of micafungin plus posaconazole or plus voriconazole was investigated herein. Methods: Scedosporium boydii (n¼17) and Scedosporium apiospermum (n¼ 26) were tested using the chequerboard method according to CLSI M38-A2 guidelines and the fractional inhibitory concentration index (FICI) was evaluated. In vivo outcome of micafungin plus posaconazole or micafungin plus voriconazole against two isolates of each of the mentioned species was evaluated in a well-established, immunocompromised, haematogenous murine model of systemic scedosporiosis. Survival and tissue burden in kidneys and brain were investigated. Results: The FICI category of ‘no interaction’ was most frequent, while ‘synergism’ or ‘antagonism’ was rarely observed. FICI failed to predict the in vivo outcome of both combinatorial treatment strategies. In vivo outcome was strain-dependent rather than species-dependent, even though effects on fungal tissue burden were more pronounced for S. boydii. Both combinations improved survival significantly when compared with untreated controls and micafungin monotherapy. Voriconazole and posaconazole did not differ in their efficacy and micafungin failed to be effective. Combinations were by trend better than voriconazole and posaconazole as single therapy, but statistically significant differences were lacking. Conclusions: No benefit of the azole/echinocandin combination was found when compared with voriconazole and posaconazole monotherapies. FICI failed to predict the outcome of in vivo drug combinations in the murine study. Keywords: combined therapy, posaconazole, voriconazole, Pseudallescheria, murine model
Introduction Scedosporium species cause a wide spectrum of infections in healthy and immunocompromised patients ranging from classical subcutaneous infections, like mycetoma, which spread via the lymphatic system, to disseminated infections with CNS involvement.1 – 3 The three major clinically relevant species within the genus Scedosporium are Scedosporium apiospermum, Scedosporium aurantiacum and Scedosporium boydii.4 – 6 While S. boydii and S. apiospermum are mainly reported from temperate climate zones, S. aurantiacum has its niche in arid areas.6 Due to the slow growth on routine media (.14 days), and the absence of standardized diagnostics assays, their real incidence and clinical
significance are likely to be underestimated.2,7 So far, S. apiospermum and S. boydii share highly similar in vitro antifungal susceptibility patterns.8,9 The majority of S. apiospermum and S. boydii isolates are resistant to amphotericin B, caspofungin, anidulafungin, isavuconazole and itraconazole.8,9 Among the azoles, voriconazole and posaconazole show the highest in vitro activity, while micafungin is the most active echinocandin.9 Due to limited treatment options at hand and high rates of therapeutic failures (including voriconazole as first-line recommendation) observed (50% – 70%),7,10 new strategies are desperately needed to improve patient outcome. A combination of a triazole and micafungin seems to be an attractive treatment regimen, as both drugs differ in their antifungal targets and subsequently in the mode of action. Such combination could act in a complementary
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Materials and methods Strain set A total of 43 Scedosporium strains (17 S. boydii and 26 S. apiospermum) were investigated (see Table 1) and identified to species level in a previous study using the amplified fragment length polymorphism method.9
In vitro susceptibility testing Strains were cultured on potato dextrose agar for 10 days at 308C. To evaluate the in vitro activity of micafungin and posaconazole or voriconazole, a two-dimensional chequerboard method according to CLSI M38-A220 and Dannaoui et al.21 was used; in addition, we applied the commercially available Etestw system (BioMe´rieux S.A, France). The MICs of azoles (posaconazole and voriconazole) and minimal effective concentrations (MECs) of micafungin were determined according to CLSI broth microdilution standards for filamentous fungi.20 For micafungin/triazole combinations, the endpoint was based on the MEC of the echinocandins and the MIC-2 (defined as an 50% reduction in growth compared with the control) of the triazoles, as the suitability of this approach was shown previously by Calvo et al. 22 Using the Etestw, MICs were determined according to the manufacturer’s instructions. The in vitro susceptibility testing was performed once for each strain and Aspergillus fumigatus ATCCw MYA 3626TM and Paecilomyces variotii ATCCw MYA 3630TM were utilized as quality controls. Combinations were evaluated by calculating the FICI [S FICI¼(A/MICdrugA +B/MICdrugB)], where A and B are the concentrations of the drugs A and B in combination and MICdrugA and MICdrugB are the MICs of the single drugs A and B. Interpretation of the FICI is as follows: ≤0.5, synergism; 0.5– 4, no interaction; and .4.0, antagonism.21,23 In a pilot study, Etestw and the chequerboard method were compared for their suitability (the criteria being operator-independent objectivity, reproducibility of results, number of unreadable or unclear results) using a randomly chosen collection of nine strains (Figure S1, available as Supplementary data at JAC Online). Strains and drug combinations were tested in triplicate and results were read by two independent investigators.
Murine model To evaluate the in vivo efficacy of micafungin and voriconazole or posaconazole, two strains per species (Table S1, available as Supplementary data at JAC Online) based on their FICI values were selected, and inocula were prepared as previously described by Calvo et al. 24 Four-week-old, male OF-1 mice (Charles River, Criffa SA, Barcelona, Spain) with a body weight between 28.0 and 30.0 g were used as model organisms. Animals were housed in standard boxes with corncob bedding and free access to food and water/grapefruit juice. Procedures were
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supervised and approved by Luis Loriente Sanz (ID 39671243) of the Veterinary and Animal Welfare Advisory Board of the Universitat Rovira i Virgili Animal Welfare and Ethics Committee (Reus, Spain). Animals were cared for in line with national guidance. The mice were randomly chosen for the different treatment and control groups. Three days prior to infection (day 23), mice in the voriconazole group (applicable for mono- and combination therapy) received grapefruit juice instead of water.25 At day 21, all mice were immunosuppressed via intraperitoneal administration of a single dose of 200 mg/kg of cyclophosphamide plus a single dose of 150 mg/kg of 5-fluorouracil given intravenously.26 Animals were infected at day 0 with 5×103 conidia/mouse via lateral tail vein injection; treatment started at day +1 and was continued until day +11. Clinical formulations of micafungin (Mycaminew), posaconazole (Noxafilw) and voriconazole (Vfendw) were used throughout. Each treatment group received micafungin at 10 mg/kg intraperitoneally once daily,27 posaconazole at 20 mg/kg orally by gavage twice a day,28 voriconazole at 40 mg/kg orally by gavage once daily,10 micafungin plus posaconazole or micafungin plus voriconazole, or served as an untreated control. For survival studies, mice were checked once a day until day +30. Animals meeting the criteria for discomfort or survivors after the experiment were killed by CO2 inhalation. The fungal load was determined on day +6 (day when the first mouse of the control group died) for comparison with the controls. Kidneys and brain were aseptically removed and approximately half of each organ was weighed, mechanically homogenized in 1 mL of 0.9% NaCl and serially 10-fold diluted. Dilutions were plated on potato dextrose agar and incubated at 308C for 7 days for cfu determination per gram of organ.
Statistical analyses Mean survival time was estimated using the Kaplan – Meier method and compared among groups with the log rank test. Tissue load was analysed with the Mann–Whitney U-test using GraphPad Prism version 6. Due to the multiple groups for comparison, a P value ,0.016 was regarded as statically significant.
Results and discussion For reasons of practicability and objectivity, chequerboard broth microdilution was the method of choice, as Etestw determination of MECs for micafungin was highly operator-dependent and difficult to read (Figure S1). Data are given in Table S2 (available as Supplementary data at JAC Online). Also, Mukherjee et al.29 report contradictory results between Etestw and the broth microdilution method when evaluating various drug combinations (including azole/echinocandin combinations) against a wide range of fungal pathogens. For both combinations tested, voriconazole/micafungin and posaconazole/micafungin, the majority of strains showed no interactions (approx. 86% and 88%, respectively; Table S3, available as Supplementary data at JAC Online). Synergistic or antagonistic effects were detected in only a few strains (Table 1). So far, our in vitro results were in agreement with findings previously published by Cuenca-Estrella et al.,30 reporting ‘no interaction’ as the most frequently observed category. In general, combinatorial in vitro data are scarce for the S. apiospermum complex; only a few data on colistin/voriconazole, colistin/caspofungin, colistin/ amphotericin B,31 micafungin/amphotericin B32 and micafungin/ voriconazole33 are available. Our study represents the first in vivo evaluation of micafungin plus voriconazole or posaconazole for members of the S. apiospermum complex. In general, all drugs applied (with the exception of
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manner and thereby improve efficacy, which has been demonstrated for other fungal pathogens.11 – 16 However, the usefulness of combination therapy for treating fungal infections is at present controversial; in most cases it is applied as salvage therapy in patients who are refractory to antifungal monotherapy.17 – 19 The aims of this study were: (i) to evaluate the efficacy of micafungin in combination with posaconazole or voriconazole against S. apiospermum and S. boydii infections in a well-established, immunocompromised murine model of systemic scedosporiosis; and (ii) to estimate whether the fractional inhibitory concentration index (FICI; in vitro characteristic measure for combination therapies) is a good parameter to predict the outcome of in vivo drug combinations. The current study represents the first in vivo study of an azole/echinocandin combination therapy against S. apiospermum complex infections.
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Table 1. In vitro activity (determined once) of voriconazole (VRC), posaconazole (POS) and micafungin (MFG), and their interaction in combination (VRC/ MFG and POS/MFG) against S. apiospermun (n¼26) and S. boydii (n¼17) strains MIC/MECa Species
Strain
FICIb
VRC
POS
MFG
VRC/MFG
interaction
POS/MFG
interaction
DH 14875 CBS 116899 CBS 117419 CBS 118233 CBS 119695 CBS 119696 CBS 119701 CBS 119706 CBS 119708 CBS 835.96 HMM 084-12 HMM 095-21 HMM 095-23 HMM 98 280 III 5.10 HMM 7137.7 FMR 13015 HMM 047-79 FMR 8763 FMR 8856 FMR 8869 IHEM 14168 HMM 13.2 HMM 20 RKI 122/04 IHEM 21162
1.0 1.0 1.0 2.0 1.0 1.0 1.0 1.0 0.5 1.0 0.5 1.0 0.5 4.0 1.0 2.0 0.25 0.5 2.0 2.0 4.0 1.0 1.0 1.0 1.0 0.5
2.0 1.0 1.0 .32.0 1.0 1.0 1.0 1.0 1.0 2.0 0.5 1.0 0.5 .32.0 1.0 .32.0 0.5 1.0 .32.0 .32.0 .32.0 .32.0 .32.0 .32.0 2.0 1.0
0.125 0.016 .16.0 0.125 0.125 0.125 0.25 0.125 0.062 0.062 8.0 .16.0 .16.0 0.125 0.031 0.125 0.125 .16.0 0.125 0.25 0.125 0.031 0.125 0.125 0.125 0.125
1.014 0.18 0.50 1.014 2.00 1.25 1.028 1.014 2.00 1.014 1.24 1.50 2.00 0.51 0.27 1.007 3.00 4.50 1.014 0.528 1.03 2.00 1.014 1.014 1.014 1.24
no interaction synergy synergy no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction synergy no interaction no interaction antagonism no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction
1.5 1.50 0.09 1.25 2.00 1.25 1.5 1.25 3.00 1.5 1.24 2.12 1.50 1.01 0.15 1.024 2.24 1.12 1.014 1.014 0.53 2.00 1.014 1.007 1.25 2.00
no interaction no interaction synergy no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction synergy no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction
S. boydii
HMM 32 HMM 3234 FMR 8346 FMR 8349 FMR 8829 FMR 8839 FMR 8863 FMR 8868 IHEM 15933 FMR 13004 HMM 0223500 FMR 12741 HMM 027-07 HMM 096-75 CBS 112.59 IHEM 21168 IHEM 21170
0.5 0.5 1.0 0.5 0.5 2.0 1.0 2.0 0.5 0.5 0.125 0.5 0.25 0.5 1.0 1.0 1.0
0.5 1.0 4.0 1.0 1.0 .32.0 1.0 .32.0 .32.0 1.0 0.25 0.5 0.25 0.5 1.0 1.0 1.0
0.125 0.062 0.125 0.125 0.125 0.25 0.062 0.5 0.125 .16.0 0.125 .16.0 0.125 .16.0 .16.0 0.5 0.125
1.24 1.50 1.014 0.74 1.5 2.00 2.00 1.01 1.5 8.24 1.01 0.48 1.24 1.24 0.54 1.007 1.007
no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction antagonism no interaction synergy no interaction no interaction no interaction no interaction no interaction
1.24 1.12 1.5 1.25 2.00 2.00 1.12 1.01 1.028 0.74 0.49 0.37 8.24 1.12 0.75 1.5 1.5
no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction synergy synergy antagonism no interaction no interaction no interaction no interaction
Culture collections: IHEM, Mycology Laboratory of the Scientific Institute of Public Health (Belgium); DH, part of the CBS (Fungal Biodiversity Centre, Utrecht, The Netherlands); FMR, Facultat d’ Medicine Reus (Spain); HMM, collection of the Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Austria. a MICs of azoles and MECs of micafungin were determined using CLSI M38-A2. b FICI interpretation: ≤0.5, synergism; .0.5– 4, no interaction; and .4, antagonism.
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60 40 20 0
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Figure 1. Cumulative mortality of mice infected with 5×103 conidia/animal of S. apiospermum (a and b) or S. boydii (c and d). aP,0.016 versus control. b P,0.016 versus micafungin. MFG, micafungin; POS, posaconazole; VRC, voriconazole.
micafungin monotherapy) prolonged survival significantly when compared with controls (P,0.016) (Figure 1). In contrast, statistically significant differences in survival were lacking between combinations and triazole monotherapies applied. Furthermore, differences in in vivo outcome as predicted by FICI were not observed, even though FICI values ranged from 0.37 to 8.24 (synergism to strong antagonism) for S. boydii and 0.09 to 4.5 (synergism to no interaction) for S. apiospermum, respectively. In particular, strains showing in vitro antagonism or no interaction for micafungin/voriconazole or micafungin/posaconazole responded equally well in vivo to strains showing in vitro synergism. Efforts were made to standardize in vitro and in vivo methods for testing the antifungal activity of combinations, but solid agreement on the in vitro method of choice was not achieved.29,34 Mice infected with the strain CBS 117419 (S. boydii) had the highest mortality rates; only voriconazole monotherapy and voriconazole plus micafungin showed some activity. With respect to survival, voriconazole and its combination were the most efficient therapies for all infected groups independent of the species and the strain tested. The effectiveness of voriconazole against scedosporiosis as monotherapy was previously demonstrated in in vivo studies.8,28,33,35 Results of fungal tissue burdens are given in Figure 2. In the treatment of S. apiospermum (FMR 13015), any combination significantly reduced the fungal load in all organs with respect to control and micafungin monotherapy, but not when compared with triazole monotherapy. For mice challenged with either of the S. boydii strains (FMR 12741 and FMR 13004), both combinations (voriconazole/micafungin and posaconazole/micafungin) significantly reduced tissue loads when compared with their
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respective monotherapies (P, 0.016). These findings were independent of the FICIs. Even though in vitro results showed an antagonistic effect for S. boydii strain FMR 13004, there was no correlation between in vitro and in vivo results. In summary, no signs of antagonism were observed in vivo either in prolongation of survival or in reduction of fungal load. Thus, the current data suggest an absence of harm or negative interaction when an azole is combined with micafungin. Rojas et al. 17 confirmed that the combination of caspofungin/voriconazole was well tolerated and that antagonism was not observed in patients. Moreover, they found caspofungin plus voriconazole to be favourable for the treatment of patients suffering from invasive fungal infections, including invasive scedosporiosis.17 Other authors reported favourable outcomes or at least the use of such a combination for the treatment of invasive scedosporiosis.17 – 19 In our study, in mice infected with either of the two S. boydii strains, posaconazole followed by voriconazole was the most effective drug treatment with respect to fungal burden. However, differences between posaconazole and voriconazole treatment were not statistically significant. Our study displays some limitations as we tested only a limited number of strains for in vivo outcome and did not perform investigations on drug –drug interactions in terms of toxicities in mice; nevertheless, our results clearly show that FICI values are not predictive for in vivo outcome and that treatment response is rather strain- than species-dependent. The discrepancy between in vitro and in vivo results, as well as the lack of species dependency on outcome, complicates any treatment prognosis. Drug efficacy experiments showed voriconazole, posaconazole and their combinations with micafungin to be the most effective treatment
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5 4 3 2 1 FG b M a, C/ FG VR / M S PO a,b C VR a,b S PO FG l b M tro a, n G Co MF a,b C/ G VR /MF S PO a,b C VR a,b S PO FG l M tro n Co
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e
d,
b,
a,
FG b,d M a, C/ FG VR /M S PO b C VR a S PO ,d FG l ,c M tro a,b ,d n G ,c Co MF a,b C/ G VR /MF S PO C VR S PO FG l M tro n Co
Figure 2. Effects of the antifungal treatments on the colony counts of the brain and kidneys of mice infected with 5×103 conidia/animal of S. apiospermum (a and b) or S. boydii (c and d). Mice were treated with: micafungin at 10 mg/kg once a day; posaconazole at 20 mg/kg twice a day; voriconazole at 40 mg/kg once a day; voriconazole/micafungin at 40 mg/kg once a day and 10 mg/kg once a day, respectively; or posaconazole/micafungin at 20 mg/kg twice a day and 10 mg/kg once a day, respectively. aP, 0.016 versus control. bP, 0.016 versus micafungin. cP, 0.016 versus posaconazole. dP, 0.016 versus voriconazole. eP,0.016 versus posaconazole/micafungin. MFG, micafungin; POS, posaconazole; VRC, voriconazole.
strategies for invasive scedosporiosis. In a few cases, Rojas et al.17 showed patients failing with treatment with voriconazole or posaconazole to benefit from combination therapy.
Funding This study was supported by internal funding.
Conclusions Voriconazole and posaconazole monotherapies were equally efficient as the combination therapies with micafungin. Therefore, combination therapy cannot be regarded as beneficial for the management of patients suffering from invasive scedosporiosis. Micafungin as monotherapy failed to be efficient. FICI-predicted antagonistic effects for posaconazole/micafungin and voriconazole/micafungin remained unconfirmed in vivo. FICI is not a reliable measure for predicting the in vivo azole/echinocandin outcome in the case of invasive scedosporiosis.
Acknowledgements We thank Bettina Sartori (Innsbruck Medical University) and Carme´ San Martin (Facultat d’ Medicina i Cie`ncies de la Salut, Universitat Rovira i Virgili) for their technical support.
Transparency declarations C. L.-F. has received grant support from the Austrian Science Fund, MFF Tirol, Astellas Pharma, Gilead Sciences, Pfizer, Schering-Plough and Merck Sharp & Dohme, has been an advisor/consultant to Gilead Sciences, Merck Sharp & Dohme, Pfizer and Schering-Plough, has received travel/accommodation expenses from Gilead Sciences, Merck Sharp & Dohme, Pfizer, Astellas and Schering-Plough, and has been paid for talks on behalf of Gilead Sciences, Merck Sharp & Dohme, Pfizer, Astellas and Schering-Plough. All other authors: none to declare.
Supplementary data Figure S1 and Tables S1 to S3 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
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6 Harun A, Gilgado F, Chen SC et al. Abundance of Pseudallescheria/ Scedosporium species in the Australian urban environment suggests a possible source for scedosporiosis including the colonization of airways in cystic fibrosis. Med Mycol 2010; 48: 70–6. 7 Tortorano AM, Richardson M, Roilides E et al. ESCMID and ECMM joint guidelines on diagnosis and management of hyalohyphomycosis: Fusarium spp., Scedosporium spp. and others. Clin Microbiol Infect 2014; 20 Suppl 3: 27 –46. 8 Gilgado F, Serena C, Cano J et al. Antifungal susceptibilities of the species of the Pseudallescheria boydii complex. Antimicrob Agents Chemother 2006; 50: 4211– 3. 9 Lackner M, de Hoog GS, Verweij PE et al. Species-specific antifungal susceptibility patterns of Scedosporium and Pseudallescheria species. Antimicrob Agents Chemother 2012; 56: 2635– 42. 10 Troke P, Aguirrebengoa K, Arteaga C et al. Treatment of scedosporiosis with voriconazole: clinical experience with 107 patients. Antimicrob Agents Chemother 2008; 52: 1743– 50. 11 Loyse A, Bicanic T, Jarvis JN. Combination antifungal therapy for cryptococcal meningitis. New Engl J Med 2013; 368: 1291 –302. 12 Gubler C, Wildi SM, Imhof A et al. Disseminated invasive aspergillosis with cerebral involvement successfully treated with caspofungin and voriconazole. Infection 2007; 35: 364–6. 13 Candoni A, Caira M, Cesaro S et al. Multicentre surveillance study on feasibility, safety and efficacy of antifungal combination therapy for proven or probable invasive fungal diseases in haematological patients: the SEIFEM real-life combo study. Mycoses 2014; 57: 342–50. 14 Seyedmousavi S, Bru¨ggemann RJ, Melchers WJ et al. Efficacy and pharmacodynamics of voriconazole combined with anidulafungin in azole-resistant invasive aspergillosis. J Antimicrob Chemother 2013; 68: 385–93. 15 Chen YL, Lehman VN, Averette A et al. Posaconazole exhibits in vitro and in vivo synergistic antifungal activity with caspofungin or FK506 against Candida albicans. PLoS One 2013; 8: e57672. 16 Silva EG, Paula CR, de Assis Baroni F et al. Voriconazole, combined with amphotericin B, in the treatment for pulmonary cryptococcosis caused by C. neoformans (serotype A) in mice with severe combined immunodeficiency (SCID). Mycopathologia 2012; 173: 445– 9. 17 Rojas R, Molina JR, Jarque I et al. Outcome of antifungal combination therapy for invasive mold infections in hematological patients is independent of the chosen combination. Mediterr J Hematol Infect Dis 2012; 4: e2012011.
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23 Johnson MD, MacDougall C, Ostrosky-Zeichner L et al. Combination antifungal therapy. Antimicrob Agents Chemother 2004; 48: 693–715. 24 Calvo E, Pastor FJ, Guarro J. Antifungal therapies in murine disseminated phaeohyphomycoses caused by Exophiala species. J Antimicrob Chemother 2010; 65: 1455– 9. 25 Graybill JR, Najvar LK, Gonzalez GM. Improving the mouse model for studying the efficacy of voriconazole. J Antimicrob Chemother 2003; 51: 1373– 6. 26 Ortoneda M, Capilla J, Pastor FJ et al. 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. 27 Rodrı´guez MM, Pastor FJ, Calvo E et al. 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. 28 Rodrı´guez MM, Pastor FJ, Salas V et al. Experimental murine scedosporiosis: histopathology and azole treatment. Antimicrob Agents Chemother 2010; 54: 3980– 4. 29 Mukherjee PK, Sheehan DJ, Hitchcock CA et al. Combination treatment of invasive fungal infections. Clin Microbiol Rev 2005; 18: 163–94. 30 Cuenca-Estrella M, Alastruey-Izquierdo A, Alcazar-Fuoli L et al. In vitro activities of 35 double combinations of antifungal agents against Scedosporium apiospermum and Scedosporium prolificans. Antimicrob Agents Chemother 2008; 52: 1136 –9. 31 Schemuth H, Dittmer S, Lackner M et al. In vitro activity of colistin as single agent and in combination with antifungals against filamentous fungi occurring in patients with cystic fibrosis. Mycoses 2013; 56: 297–303. 32 Yustes C, Guarro J. In vitro synergistic interaction between amphotericin B and micafungin against Scedosporium spp. Antimicrob Agents Chemother 2005; 49: 3498– 500. 33 Rodrı´guez MM, Calvo E, Serena C et al. Effects of double and triple combinations of antifungal drugs in a murine model of disseminated infection by Scedosporium prolificans. Antimicrob Agents Chemother 2009; 53: 2153– 5. 34 Vazquez JA. Clinical practice: combination antifungal therapy for mold infections: much ado about nothing? Clin Infect Dis 2008; 46: 1889–901. 35 Capilla J, Serena C, Pastor FJ et al. Efficacy of voriconazole in treatment of systemic scedosporiosis in neutropenic mice. Antimicrob Agents Chemother 2003; 47: 3976– 8.
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2 Cortez KJ, Roilides E, Quiroz-Telles F et al. Infections caused by Scedosporium spp. Clin Microbiol Rev 2008; 21: 157– 97.
19 Beier F, Kittan N, Holzmann T et al. Successful treatment of Scedosporium apiospermum soft tissue abscess with caspofungin and voriconazole in a severely immunocompromised patient with acute myeloid leukemia. Transpl Infect Dis 2010; 12: 538– 42.
J Antimicrob Chemother doi:10.1093/jac/dku224
Assessing micafungin/triazole combinations for the treatment of invasive scedosporiosis due to Scedosporium apiospermum and Scedosporium boydii Michaela Lackner1*†, Fabiola Ferna´ndez-Silva2†, Josep Guarro3 and Cornelia Lass-Flo¨rl1 1 Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria; 2Unitat d’ Anatomia Patolo`gica, Facultat d’ Medicina i Cie`ncies de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain; 3Unitat d’ Microbiologia, Facultat d’ Medicina i Cie`ncies de la Salut, IISPV, Universitat Rovira i Virgili, Reus, Spain
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*Corresponding author. Tel: +43-(0)512-9003-70725; Fax: +43-(0)512-9003-73700; E-mail: [email protected] †Both authors contributed equally and share the first author position.
Received 15 April 2014; returned 3 May 2014; revised 13 May 2014; accepted 23 May 2014 Objectives: Scedosporium infections are associated with high therapeutic failure rates. Combination therapy may be an alternative approach to improve outcome. The in vitro and in vivo efficacy of micafungin plus posaconazole or plus voriconazole was investigated herein. Methods: Scedosporium boydii (n¼17) and Scedosporium apiospermum (n¼ 26) were tested using the chequerboard method according to CLSI M38-A2 guidelines and the fractional inhibitory concentration index (FICI) was evaluated. In vivo outcome of micafungin plus posaconazole or micafungin plus voriconazole against two isolates of each of the mentioned species was evaluated in a well-established, immunocompromised, haematogenous murine model of systemic scedosporiosis. Survival and tissue burden in kidneys and brain were investigated. Results: The FICI category of ‘no interaction’ was most frequent, while ‘synergism’ or ‘antagonism’ was rarely observed. FICI failed to predict the in vivo outcome of both combinatorial treatment strategies. In vivo outcome was strain-dependent rather than species-dependent, even though effects on fungal tissue burden were more pronounced for S. boydii. Both combinations improved survival significantly when compared with untreated controls and micafungin monotherapy. Voriconazole and posaconazole did not differ in their efficacy and micafungin failed to be effective. Combinations were by trend better than voriconazole and posaconazole as single therapy, but statistically significant differences were lacking. Conclusions: No benefit of the azole/echinocandin combination was found when compared with voriconazole and posaconazole monotherapies. FICI failed to predict the outcome of in vivo drug combinations in the murine study. Keywords: combined therapy, posaconazole, voriconazole, Pseudallescheria, murine model
Introduction Scedosporium species cause a wide spectrum of infections in healthy and immunocompromised patients ranging from classical subcutaneous infections, like mycetoma, which spread via the lymphatic system, to disseminated infections with CNS involvement.1 – 3 The three major clinically relevant species within the genus Scedosporium are Scedosporium apiospermum, Scedosporium aurantiacum and Scedosporium boydii.4 – 6 While S. boydii and S. apiospermum are mainly reported from temperate climate zones, S. aurantiacum has its niche in arid areas.6 Due to the slow growth on routine media (.14 days), and the absence of standardized diagnostics assays, their real incidence and clinical
significance are likely to be underestimated.2,7 So far, S. apiospermum and S. boydii share highly similar in vitro antifungal susceptibility patterns.8,9 The majority of S. apiospermum and S. boydii isolates are resistant to amphotericin B, caspofungin, anidulafungin, isavuconazole and itraconazole.8,9 Among the azoles, voriconazole and posaconazole show the highest in vitro activity, while micafungin is the most active echinocandin.9 Due to limited treatment options at hand and high rates of therapeutic failures (including voriconazole as first-line recommendation) observed (50% – 70%),7,10 new strategies are desperately needed to improve patient outcome. A combination of a triazole and micafungin seems to be an attractive treatment regimen, as both drugs differ in their antifungal targets and subsequently in the mode of action. Such combination could act in a complementary
# The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
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Materials and methods Strain set A total of 43 Scedosporium strains (17 S. boydii and 26 S. apiospermum) were investigated (see Table 1) and identified to species level in a previous study using the amplified fragment length polymorphism method.9
In vitro susceptibility testing Strains were cultured on potato dextrose agar for 10 days at 308C. To evaluate the in vitro activity of micafungin and posaconazole or voriconazole, a two-dimensional chequerboard method according to CLSI M38-A220 and Dannaoui et al.21 was used; in addition, we applied the commercially available Etestw system (BioMe´rieux S.A, France). The MICs of azoles (posaconazole and voriconazole) and minimal effective concentrations (MECs) of micafungin were determined according to CLSI broth microdilution standards for filamentous fungi.20 For micafungin/triazole combinations, the endpoint was based on the MEC of the echinocandins and the MIC-2 (defined as an 50% reduction in growth compared with the control) of the triazoles, as the suitability of this approach was shown previously by Calvo et al. 22 Using the Etestw, MICs were determined according to the manufacturer’s instructions. The in vitro susceptibility testing was performed once for each strain and Aspergillus fumigatus ATCCw MYA 3626TM and Paecilomyces variotii ATCCw MYA 3630TM were utilized as quality controls. Combinations were evaluated by calculating the FICI [S FICI¼(A/MICdrugA +B/MICdrugB)], where A and B are the concentrations of the drugs A and B in combination and MICdrugA and MICdrugB are the MICs of the single drugs A and B. Interpretation of the FICI is as follows: ≤0.5, synergism; 0.5– 4, no interaction; and .4.0, antagonism.21,23 In a pilot study, Etestw and the chequerboard method were compared for their suitability (the criteria being operator-independent objectivity, reproducibility of results, number of unreadable or unclear results) using a randomly chosen collection of nine strains (Figure S1, available as Supplementary data at JAC Online). Strains and drug combinations were tested in triplicate and results were read by two independent investigators.
Murine model To evaluate the in vivo efficacy of micafungin and voriconazole or posaconazole, two strains per species (Table S1, available as Supplementary data at JAC Online) based on their FICI values were selected, and inocula were prepared as previously described by Calvo et al. 24 Four-week-old, male OF-1 mice (Charles River, Criffa SA, Barcelona, Spain) with a body weight between 28.0 and 30.0 g were used as model organisms. Animals were housed in standard boxes with corncob bedding and free access to food and water/grapefruit juice. Procedures were
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supervised and approved by Luis Loriente Sanz (ID 39671243) of the Veterinary and Animal Welfare Advisory Board of the Universitat Rovira i Virgili Animal Welfare and Ethics Committee (Reus, Spain). Animals were cared for in line with national guidance. The mice were randomly chosen for the different treatment and control groups. Three days prior to infection (day 23), mice in the voriconazole group (applicable for mono- and combination therapy) received grapefruit juice instead of water.25 At day 21, all mice were immunosuppressed via intraperitoneal administration of a single dose of 200 mg/kg of cyclophosphamide plus a single dose of 150 mg/kg of 5-fluorouracil given intravenously.26 Animals were infected at day 0 with 5×103 conidia/mouse via lateral tail vein injection; treatment started at day +1 and was continued until day +11. Clinical formulations of micafungin (Mycaminew), posaconazole (Noxafilw) and voriconazole (Vfendw) were used throughout. Each treatment group received micafungin at 10 mg/kg intraperitoneally once daily,27 posaconazole at 20 mg/kg orally by gavage twice a day,28 voriconazole at 40 mg/kg orally by gavage once daily,10 micafungin plus posaconazole or micafungin plus voriconazole, or served as an untreated control. For survival studies, mice were checked once a day until day +30. Animals meeting the criteria for discomfort or survivors after the experiment were killed by CO2 inhalation. The fungal load was determined on day +6 (day when the first mouse of the control group died) for comparison with the controls. Kidneys and brain were aseptically removed and approximately half of each organ was weighed, mechanically homogenized in 1 mL of 0.9% NaCl and serially 10-fold diluted. Dilutions were plated on potato dextrose agar and incubated at 308C for 7 days for cfu determination per gram of organ.
Statistical analyses Mean survival time was estimated using the Kaplan – Meier method and compared among groups with the log rank test. Tissue load was analysed with the Mann–Whitney U-test using GraphPad Prism version 6. Due to the multiple groups for comparison, a P value ,0.016 was regarded as statically significant.
Results and discussion For reasons of practicability and objectivity, chequerboard broth microdilution was the method of choice, as Etestw determination of MECs for micafungin was highly operator-dependent and difficult to read (Figure S1). Data are given in Table S2 (available as Supplementary data at JAC Online). Also, Mukherjee et al.29 report contradictory results between Etestw and the broth microdilution method when evaluating various drug combinations (including azole/echinocandin combinations) against a wide range of fungal pathogens. For both combinations tested, voriconazole/micafungin and posaconazole/micafungin, the majority of strains showed no interactions (approx. 86% and 88%, respectively; Table S3, available as Supplementary data at JAC Online). Synergistic or antagonistic effects were detected in only a few strains (Table 1). So far, our in vitro results were in agreement with findings previously published by Cuenca-Estrella et al.,30 reporting ‘no interaction’ as the most frequently observed category. In general, combinatorial in vitro data are scarce for the S. apiospermum complex; only a few data on colistin/voriconazole, colistin/caspofungin, colistin/ amphotericin B,31 micafungin/amphotericin B32 and micafungin/ voriconazole33 are available. Our study represents the first in vivo evaluation of micafungin plus voriconazole or posaconazole for members of the S. apiospermum complex. In general, all drugs applied (with the exception of
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manner and thereby improve efficacy, which has been demonstrated for other fungal pathogens.11 – 16 However, the usefulness of combination therapy for treating fungal infections is at present controversial; in most cases it is applied as salvage therapy in patients who are refractory to antifungal monotherapy.17 – 19 The aims of this study were: (i) to evaluate the efficacy of micafungin in combination with posaconazole or voriconazole against S. apiospermum and S. boydii infections in a well-established, immunocompromised murine model of systemic scedosporiosis; and (ii) to estimate whether the fractional inhibitory concentration index (FICI; in vitro characteristic measure for combination therapies) is a good parameter to predict the outcome of in vivo drug combinations. The current study represents the first in vivo study of an azole/echinocandin combination therapy against S. apiospermum complex infections.
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Table 1. In vitro activity (determined once) of voriconazole (VRC), posaconazole (POS) and micafungin (MFG), and their interaction in combination (VRC/ MFG and POS/MFG) against S. apiospermun (n¼26) and S. boydii (n¼17) strains MIC/MECa Species
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POS
MFG
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interaction
DH 14875 CBS 116899 CBS 117419 CBS 118233 CBS 119695 CBS 119696 CBS 119701 CBS 119706 CBS 119708 CBS 835.96 HMM 084-12 HMM 095-21 HMM 095-23 HMM 98 280 III 5.10 HMM 7137.7 FMR 13015 HMM 047-79 FMR 8763 FMR 8856 FMR 8869 IHEM 14168 HMM 13.2 HMM 20 RKI 122/04 IHEM 21162
1.0 1.0 1.0 2.0 1.0 1.0 1.0 1.0 0.5 1.0 0.5 1.0 0.5 4.0 1.0 2.0 0.25 0.5 2.0 2.0 4.0 1.0 1.0 1.0 1.0 0.5
2.0 1.0 1.0 .32.0 1.0 1.0 1.0 1.0 1.0 2.0 0.5 1.0 0.5 .32.0 1.0 .32.0 0.5 1.0 .32.0 .32.0 .32.0 .32.0 .32.0 .32.0 2.0 1.0
0.125 0.016 .16.0 0.125 0.125 0.125 0.25 0.125 0.062 0.062 8.0 .16.0 .16.0 0.125 0.031 0.125 0.125 .16.0 0.125 0.25 0.125 0.031 0.125 0.125 0.125 0.125
1.014 0.18 0.50 1.014 2.00 1.25 1.028 1.014 2.00 1.014 1.24 1.50 2.00 0.51 0.27 1.007 3.00 4.50 1.014 0.528 1.03 2.00 1.014 1.014 1.014 1.24
no interaction synergy synergy no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction synergy no interaction no interaction antagonism no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction
1.5 1.50 0.09 1.25 2.00 1.25 1.5 1.25 3.00 1.5 1.24 2.12 1.50 1.01 0.15 1.024 2.24 1.12 1.014 1.014 0.53 2.00 1.014 1.007 1.25 2.00
no interaction no interaction synergy no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction synergy no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction
S. boydii
HMM 32 HMM 3234 FMR 8346 FMR 8349 FMR 8829 FMR 8839 FMR 8863 FMR 8868 IHEM 15933 FMR 13004 HMM 0223500 FMR 12741 HMM 027-07 HMM 096-75 CBS 112.59 IHEM 21168 IHEM 21170
0.5 0.5 1.0 0.5 0.5 2.0 1.0 2.0 0.5 0.5 0.125 0.5 0.25 0.5 1.0 1.0 1.0
0.5 1.0 4.0 1.0 1.0 .32.0 1.0 .32.0 .32.0 1.0 0.25 0.5 0.25 0.5 1.0 1.0 1.0
0.125 0.062 0.125 0.125 0.125 0.25 0.062 0.5 0.125 .16.0 0.125 .16.0 0.125 .16.0 .16.0 0.5 0.125
1.24 1.50 1.014 0.74 1.5 2.00 2.00 1.01 1.5 8.24 1.01 0.48 1.24 1.24 0.54 1.007 1.007
no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction antagonism no interaction synergy no interaction no interaction no interaction no interaction no interaction
1.24 1.12 1.5 1.25 2.00 2.00 1.12 1.01 1.028 0.74 0.49 0.37 8.24 1.12 0.75 1.5 1.5
no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction no interaction synergy synergy antagonism no interaction no interaction no interaction no interaction
Culture collections: IHEM, Mycology Laboratory of the Scientific Institute of Public Health (Belgium); DH, part of the CBS (Fungal Biodiversity Centre, Utrecht, The Netherlands); FMR, Facultat d’ Medicine Reus (Spain); HMM, collection of the Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Austria. a MICs of azoles and MECs of micafungin were determined using CLSI M38-A2. b FICI interpretation: ≤0.5, synergism; .0.5– 4, no interaction; and .4, antagonism.
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Figure 1. Cumulative mortality of mice infected with 5×103 conidia/animal of S. apiospermum (a and b) or S. boydii (c and d). aP,0.016 versus control. b P,0.016 versus micafungin. MFG, micafungin; POS, posaconazole; VRC, voriconazole.
micafungin monotherapy) prolonged survival significantly when compared with controls (P,0.016) (Figure 1). In contrast, statistically significant differences in survival were lacking between combinations and triazole monotherapies applied. Furthermore, differences in in vivo outcome as predicted by FICI were not observed, even though FICI values ranged from 0.37 to 8.24 (synergism to strong antagonism) for S. boydii and 0.09 to 4.5 (synergism to no interaction) for S. apiospermum, respectively. In particular, strains showing in vitro antagonism or no interaction for micafungin/voriconazole or micafungin/posaconazole responded equally well in vivo to strains showing in vitro synergism. Efforts were made to standardize in vitro and in vivo methods for testing the antifungal activity of combinations, but solid agreement on the in vitro method of choice was not achieved.29,34 Mice infected with the strain CBS 117419 (S. boydii) had the highest mortality rates; only voriconazole monotherapy and voriconazole plus micafungin showed some activity. With respect to survival, voriconazole and its combination were the most efficient therapies for all infected groups independent of the species and the strain tested. The effectiveness of voriconazole against scedosporiosis as monotherapy was previously demonstrated in in vivo studies.8,28,33,35 Results of fungal tissue burdens are given in Figure 2. In the treatment of S. apiospermum (FMR 13015), any combination significantly reduced the fungal load in all organs with respect to control and micafungin monotherapy, but not when compared with triazole monotherapy. For mice challenged with either of the S. boydii strains (FMR 12741 and FMR 13004), both combinations (voriconazole/micafungin and posaconazole/micafungin) significantly reduced tissue loads when compared with their
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respective monotherapies (P, 0.016). These findings were independent of the FICIs. Even though in vitro results showed an antagonistic effect for S. boydii strain FMR 13004, there was no correlation between in vitro and in vivo results. In summary, no signs of antagonism were observed in vivo either in prolongation of survival or in reduction of fungal load. Thus, the current data suggest an absence of harm or negative interaction when an azole is combined with micafungin. Rojas et al. 17 confirmed that the combination of caspofungin/voriconazole was well tolerated and that antagonism was not observed in patients. Moreover, they found caspofungin plus voriconazole to be favourable for the treatment of patients suffering from invasive fungal infections, including invasive scedosporiosis.17 Other authors reported favourable outcomes or at least the use of such a combination for the treatment of invasive scedosporiosis.17 – 19 In our study, in mice infected with either of the two S. boydii strains, posaconazole followed by voriconazole was the most effective drug treatment with respect to fungal burden. However, differences between posaconazole and voriconazole treatment were not statistically significant. Our study displays some limitations as we tested only a limited number of strains for in vivo outcome and did not perform investigations on drug –drug interactions in terms of toxicities in mice; nevertheless, our results clearly show that FICI values are not predictive for in vivo outcome and that treatment response is rather strain- than species-dependent. The discrepancy between in vitro and in vivo results, as well as the lack of species dependency on outcome, complicates any treatment prognosis. Drug efficacy experiments showed voriconazole, posaconazole and their combinations with micafungin to be the most effective treatment
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Figure 2. Effects of the antifungal treatments on the colony counts of the brain and kidneys of mice infected with 5×103 conidia/animal of S. apiospermum (a and b) or S. boydii (c and d). Mice were treated with: micafungin at 10 mg/kg once a day; posaconazole at 20 mg/kg twice a day; voriconazole at 40 mg/kg once a day; voriconazole/micafungin at 40 mg/kg once a day and 10 mg/kg once a day, respectively; or posaconazole/micafungin at 20 mg/kg twice a day and 10 mg/kg once a day, respectively. aP, 0.016 versus control. bP, 0.016 versus micafungin. cP, 0.016 versus posaconazole. dP, 0.016 versus voriconazole. eP,0.016 versus posaconazole/micafungin. MFG, micafungin; POS, posaconazole; VRC, voriconazole.
strategies for invasive scedosporiosis. In a few cases, Rojas et al.17 showed patients failing with treatment with voriconazole or posaconazole to benefit from combination therapy.
Funding This study was supported by internal funding.
Conclusions Voriconazole and posaconazole monotherapies were equally efficient as the combination therapies with micafungin. Therefore, combination therapy cannot be regarded as beneficial for the management of patients suffering from invasive scedosporiosis. Micafungin as monotherapy failed to be efficient. FICI-predicted antagonistic effects for posaconazole/micafungin and voriconazole/micafungin remained unconfirmed in vivo. FICI is not a reliable measure for predicting the in vivo azole/echinocandin outcome in the case of invasive scedosporiosis.
Acknowledgements We thank Bettina Sartori (Innsbruck Medical University) and Carme´ San Martin (Facultat d’ Medicina i Cie`ncies de la Salut, Universitat Rovira i Virgili) for their technical support.
Transparency declarations C. L.-F. has received grant support from the Austrian Science Fund, MFF Tirol, Astellas Pharma, Gilead Sciences, Pfizer, Schering-Plough and Merck Sharp & Dohme, has been an advisor/consultant to Gilead Sciences, Merck Sharp & Dohme, Pfizer and Schering-Plough, has received travel/accommodation expenses from Gilead Sciences, Merck Sharp & Dohme, Pfizer, Astellas and Schering-Plough, and has been paid for talks on behalf of Gilead Sciences, Merck Sharp & Dohme, Pfizer, Astellas and Schering-Plough. All other authors: none to declare.
Supplementary data Figure S1 and Tables S1 to S3 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
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