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In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus from different geographical regions
DMB-14555; No of Pages 3 Diagnostic Microbiology and Infectious Disease xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
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In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus from different geographical regions Hamed Fakhim a,b, Afsane Vaezi c,d, Eric Dannaoui e, Cheshta Sharma f, Bita Mousavi g, Anuradha Chowdhary f, Jacques F. Meis h,i, Hamid Badali c,j,⁎ a
Department of Medical Parasitology and Mycology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran Cellular and Molecular Research Center, Urmia University of Medical Sciences, Urmia, Iran c Department of Medical Mycology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran d Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran e Université Paris-Descartes, Faculté de Médecine, APHP, Hôpital Européen Georges Pompidou, Unité de Parasitologie-Mycologie, Service de Microbiologie, Paris, France f Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India g Dynamyc Research Group (EA 7380), Paris Est Créteil University, Ecole nationale vétérinaire d'Alfort, Créteil, France h Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital (CWZ), Nijmegen, The Netherlands i Center of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands j Pharmaceutical Sciences Research Center, Hemoglobinopathy Institute, Mazandaran University of Medical Sciences, Sari, Iran b
a r t i c l e
i n f o
Article history: Received 8 September 2017 Received in revised form 19 February 2018 Accepted 3 March 2018 Available online xxxx
a b s t r a c t In vitro interaction of voriconazole with micafungin was evaluated against 33 clinical Aspergillus fumigatus isolates, including azole-resistant (n = 31) and -susceptible (n = 2) isolates. Interaction was synergistic for only 1 resistant isolate carrying the TR34/L98H mutation. No antagonistic effects were observed for 96.8% of azole-resistant isolates. © 2018 Elsevier Inc. All rights reserved.
Keywords: In vitro interactions Voriconazole Micafungin Aspergillus fumigatus
Azole-resistant Aspergillus fumigatus isolates have emerged as a major source of life-threatening aspergillosis in hospitalized patients, and these infections have been associated with significant morbidity and mortality even when properly diagnosed and treated (Lestrade et al., 2016; Verweij et al., 2016). Azole resistance, which is reported worldwide, may be due to long-term azole therapy in patients or the selective pressure of agrochemical triazoles in the environment (Chowdhary et al., 2013; Chowdhary and Meis, 2018). A vicious cycle is created as azole-resistant aspergillosis forces us to rely on limited additional available antifungal drugs. However, combination therapy may be an interesting approach to treat patients with Aspergillus infection (Johnson et al., 2004; Marr et al., 2015; Mukherjee et al., 2005; Verweij
⁎ Corresponding author. Tel.: +98-9128413720 (Mobile); fax: +98-1133543249. E-mail addresses: [email protected] (H. Fakhim), [email protected] (A. Vaezi), [email protected] (E. Dannaoui), [email protected] (C. Sharma), [email protected] (B. Mousavi), [email protected] (A. Chowdhary), [email protected] (J.F. Meis), [email protected] (H. Badali).
et al., 2016). There is some evidence supporting combination therapy with 2 antifungals with different mechanism of action (Aliff et al., 2003; Marr et al., 2015), especially the combination of echinocandins with voriconazole (Aliff et al., 2003; Cuenca-Estrella et al., 2005; Lewis and Kontoyiannis, 2005; Marr et al., 2004; Perkhofer et al., 2008). The aim of this study was to determine the in vitro interaction of voriconazole with micafungin against azole-resistant A. fumigatus clinical isolates harboring various resistance mechanisms and originating from different geographical regions. A panel of 33 well-characterized clinical A. fumigatus strains, comprising azole-resistant [i.e., TR34/L98H (n = 10), TR46/Y121F/ T289A (n = 6), G54E (n = 8), M220 (n = 3), cyp51A wild-type gene (n = 4)] and azole-susceptible (wild type; n = 2) strains, was tested. These isolates originated from the Netherlands, Romania, Tanzania, India, and Iran. All isolates were stored at −80°C at the Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, India, and also at the reference culture collection of Invasive Fungi Research Center, Mazandaran University of Medical Sciences,
https://doi.org/10.1016/j.diagmicrobio.2018.03.003 0732-8893/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Fakhim H, et al, In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus..., Diagn Microbiol Infect Dis (2018), https://doi.org/10.1016/j.diagmicrobio.2018.03.003
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H. Fakhim et al. / Diagnostic Microbiology and Infectious Disease xxx (2018) xxx–xxx
micafungin MECs when tested alone was 0.125–N16 μg/mL and 0.002– 0.031 μg/mL, respectively. By using a complete inhibition endpoint, FICI of TR34/L98H, TR46/Y121F/T289A, G54E, M220, and isolates without cyp51A mutations ranged from 0.37 to 2.06, 0.75 to 1.5, 0.62 to 2.5, 0.5 to 1.5, and 0.62 to 1.25, respectively. The widest range of FICI-0 was found for isolates harboring the TR34/ L98H mutation. The combination exhibited indifferent activity against wild-type controls isolates. The interaction between voriconazole and micafungin was indifferent for 31 of 33 (93.9%) isolates. The combination was synergistic for only 1 isolate with TR34/L98H mutation and antagonistic for 1 azole-resistant isolate with the G54E mutation. We assessed the combination of voriconazole with micafungin against A. fumigatus harboring various azole-resistant mechanisms from different regions. In the current study, 64.5% (20 of 31) of azoleresistant strains were not susceptible to voriconazole. Although, voriconazole combined with micafungin did not exhibit a synergistic activity against 96.8% (30/31) of azole-resistant isolates, this combination against 83.9% (26/31) and 90.3% (28/31) of these isolates showed a 2- to 3-log2-dilution step lower activity than voriconazole and micafungin alone, respectively. A previous study by Seyedmousavi et al. (2013) reported a synergistic interaction of voriconazole and anidulafungin combination. However, this study also pointed out that synergistic drug interactions, based on measuring the FICI, were dependent on MIC endpoints, in which FICI was inversely related to voriconazole and anidulafungin MICs and were influenced by cyp51A genotype. Thus, voriconazole–echinocandin combination could possibly be less effective in voriconazole-resistant strains with high MICs. However, other studies
Sari, Iran. All isolates were previously identified by conventional and molecular methods, i.e., cultures supplemented with 4 μg/mL and 1 μg/mL of itraconazole and voriconazole, respectively, at 45°C for 72 h in the dark, in vitro antifungal susceptibility as described in CLSI document M38-A2 (CLSI, 2008), DNA sequencing of the partial βtubulin gene (Abastabar et al., 2016; Badali et al., 2013; Nabili et al., 2016), and sequencing of Cyp51A and promotor region (Chowdhary et al., 2015; Tamura et al., 2011). In vitro antifungal susceptibility to voriconazole (VRC; Pfizer, Groton, CT, USA) and micafungin (MFG; Astellas, Toyama, Japan) alone was determined as described in CLSI document M38-A2 (CLSI, 2008). Isolates with a voriconazole MIC of N2 μg/mL were considered resistant. The in vitro interaction between voriconazole and micafungin was tested by using a checkerboard method based on the microdilution broth reference technique (M38-A2) (CLSI, 2008; Odds, 2003). The concentration range of voriconazole (0.25 to 16 μg/mL or 0.031 to 2 μg/mL) depended on the MIC results of each isolate and was from 0.0002 to 0.125 μg/mL for micafungin. MICs/MECs were determined after 48 h of incubation at 35°C. Both complete (VisuMIC-0) and partial (VisuMIC50) inhibition endpoints were used (Arikan et al., 2002; Planche et al., 2012). The fractional inhibitory concentration index (FICI) was calculated and the interaction was interpreted as synergistic (FICI ≤ 0.5), indifferent (FICI 0.5–4), or antagonistic (FICI N 4) (Odds, 2003). Table 1 summarizes the results of in vitro interaction of voriconazole with micafungin against all A. fumigatus strains. A total of 64.5% (20 of 31) and 39.9% (13 of 33) of A. fumigatus were resistant and susceptible to voriconazole, respectively. The range of voriconazole MICs and
Table 1 In vitro interaction of voriconazole with micafungin against azole-resistant Aspergillus fumigatus isolates. Strains
Mutations
Origin
VRC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
TR46/Y121F/T289A TR46/Y121F/T289A TR46/Y121F/T289A TR46/Y121F/T289A TR46/Y121F/T289A TR46/Y121F/T289A TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H G54E G54E G54E G54E G54E G54E G54E G54E M220 M220 M220 No mutation No mutation No mutation No mutation Wild type (ATCC204305) Wild type (290/E1/11)
India India Tanzania Tanzania Netherlands Netherlands Tanzania Tanzania Romania Romania India India Netherlands Netherlands Iran Iran Romania Romania Tanzania Tanzania India India Netherlands Netherlands Netherlands Netherlands Netherlands India India Iran Iran US (Virginia) India
MICs/MECs (μg/mL) in combination
MICs/MECs (μg/mL) of the drugs alone
FICI in combination
MFG
VRC
MFG
MIC
MEC
MIC-0
MIC-50
MIC-0
MEC
MIC-0
MEC
N16 16 N16 N16 N16 N16 4 8 16 16 8 4 8 8 4 16 1 1 0.125 0.125 1 0.25 0.125 0.125 0.5 0.5 8 4 16 4 4 0.125 0.125
0.008 0.016 0.004 0.002 0.016 0.031 0.016 0.008 0.016 0.016 0.008 0.016 0.008 0.016 0.016 0.016 0.008 0.004 0.016 0.016 0.008 0.016 0.016 0.008 0.016 0.008 0.016 0.016 0.016 0.016 0.016 0.004 0.008
8 4 8 16 16 16 2 8 8 8 4 1 8 16 2 8 0.25 0.5 0.125 0.063 0.25 0.125 0.125 0.063 0.125 0.25 2 2 8 0.25 4 0.125 0.063
16 1 4 8 4 8 4 8 16 16 8 4 16 16 4 16 0.25 0.25 0.063 0.063 0.125 0.125 0.25 0.125 0.125 0. 25 8 4 16 0.25 2 0.125 0.125
0.004 0.008 0.002 0.002 0.004 0.008 0.004 0.004 0.004 0.008 0.004 0.002 0.008 0.001 0.004 0.008 0.004 0.008 0.002 0.004 0.004 0.002 0.008 0.004 0.008 0.008 0.004 0.002 0.004 0.002 0.004 0.001 0.002
0.016 0.016 0.004 0.004 0.016 0.031 0.016 0.016 0.008 0.016 0.016 0.004 0.016 0.004 0.016 0.016 0.016 0.016 0.008 0.016 0.016 0.016 0.016 0.016 0.016 0.016 0.016 0.008 0.016 0.016 0.016 0.004 0.008
0.75 0.75 0.75 1.5 0.75 0.75 0.75 1.5 0.75 1 1 0.37 2 2.06 0.75 1 0.75 2.5 1.12 0.75 0.75 0.62 1.5 1 0.75 1.5 0.5 0.62 0.75 1.12 1.25 1.25 0.75
2.5 1.06 1.12 2.25 1.12 1.25 2 3 1.5 2 3 1.25 4 2.25 2 2 2.25 4.25 1 1.5 2.12 1.5 3 3 1.25 2.5 2 2 2 2 1.5 2 2
Abbreviations: TR34/L98H and TR46/Y121F/T289A = tandem repeat and mutations (promotor duplication and cyp51A substitutions); M220 and G54E = single point mutation (cyp51A substitution); no mutation = without any mutations in cyp51A gene; VRC = voriconazole; MFG = micafungin; MICs = minimum inhibitory concentration; MECs = minimum effective concentration; MIC-50 = 50% inhibition; MIC-0 = 100% inhibition; FICI = fractional inhibitory concentration index.
Please cite this article as: Fakhim H, et al, In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus..., Diagn Microbiol Infect Dis (2018), https://doi.org/10.1016/j.diagmicrobio.2018.03.003
H. Fakhim et al. / Diagnostic Microbiology and Infectious Disease xxx (2018) xxx–xxx
reported no significant differences of the combination of echinocandins with azoles against Aspergillus species (Heyn et al., 2005; Lewis and Kontoyiannis, 2005; Perkhofer et al., 2008; Planche et al., 2012; Rubin et al., 2002). Notably, our study suggested that the different resistance mechanisms of isolates originating from different geographical regions compared to wild-type control isolates cannot influence the synergistic effects of combined agents. We were unable to find a correlation between the resistant mechanisms and in the vitro combination results. The earlier reported combination of anidulafungin with voriconazole had less effect against voriconazole-resistant strains (Seyedmousavi et al., 2013). The use of different visually determined MIC endpoints, wide ranges of FIC index cutoff values, and various methodologies employed may be important factors associated with the underlying explanation for in vitro results. In our study, we used cutoff values to indicate that the interactions were synergistic if the FIC index was ≤0.5, indifferent if the FIC index was 0.5 to 4, and antagonistic if the FIC index was N4 (Odds, 2003). Evidence to support combination therapy for azole-resistant A. fumigatus infections is scarce at present. Although in vitro interactions are conducted in well-controlled conditions with accurate concentrations of multiple drugs and known inocula of spores, it can be different from the unpredictable drug concentrations and fungal burdens in patients. In addition, in vitro studies cannot take into account the effect of the host immune system. Alternative antifungal strategies including combination therapy to avoid further emergence of azole resistance at the first suspicion of invasive aspergillosis in critical ill patients can be considered as the possible resolution for the treatment of azole-resistant aspergillosis (Martín-Peña et al., 2014). In conclusion, in vitro interaction of voriconazole and micafungin was indifferent against 93.9% of azole-resistant A. fumigatus isolates. An increased likelihood that the resistant A. fumigatus will be susceptible to at least one of the components makes this a suitable option as an empiric combination regimen (Verweij et al., 2015). More in vitro and in vivo studies are necessary to address questions regarding azole and echinocandin combination therapy. Acknowledgments This study was financially supported by a grant from the Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India. Hamed Fakhim was supported by an ESCMID observership grant (ID: 1002) which is gratefully acknowledged. Hamid Badali was funded by a grant (no. 2407) from Mazandaran University of Medical Sciences, Sari, Iran. Author contributions H.F and H.B.: research concept/design, data collection, susceptibility testing, analysis, interpretation of data, and drafting article; A.V.: susceptibility testing, analysis, interpretation of data, and drafting article; E.D.: analysis, interpretation of data, critical revision of article, and approval of article; C.S.: susceptibility testing, analysis, interpretation of data; B.M.: drafting article; A.C., J.F.M. ,and H.B.: interpretation of data, and critical revision and approval of article. All authors read and approved the final manuscript. Conflict of interest J.F.M. received grants from Astellas, Merck, and Basilea. He has been a consultant to Basilea, Scynexis, and Merck and received speaker fees from Merck, Pfizer, Gilead, TEVA, and United Medical. During the past 5 years, E.D. has received research grants from MSD and Gilead; travel grants from Gilead, MSD, Pfizer, and Astellas; and speaker's fee from Gilead, MSD, and Astellas. All other authors no potential conflicts of interest.
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Seyedmousavi S, Meletiadis J, Melchers WJ, Rijs AJ, Mouton JW, Verweij PE. In vitro interaction of voriconazole and anidulafungin against triazole-resistant Aspergillus fumigatus. Antimicrob Agents Chemother 2013;57(2):796–803. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28(10):2731–9. Verweij PE, Ananda-Rajah M, Andes D, Arendrup MC, Brüggemann RJ, Chowdhary A, et al. International expert opinion on the management of infection caused by azoleresistant Aspergillus fumigatus. Drug Resist Updat 2015;21-22:30–40. https://doi. org/10.1016/j.drup.2015.08.001. Verweij PE, Chowdhary A, Melchers WJ, Meis JF. Azole resistance in Aspergillus fumigatus: can we retain the clinical use of mold-active antifungal azoles? Clin Infect Dis 2016; 62(3):362–8. https://doi.org/10.1093/cid/civ885.
Please cite this article as: Fakhim H, et al, In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus..., Diagn Microbiol Infect Dis (2018), https://doi.org/10.1016/j.diagmicrobio.2018.03.003
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Note
In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus from different geographical regions Hamed Fakhim a,b, Afsane Vaezi c,d, Eric Dannaoui e, Cheshta Sharma f, Bita Mousavi g, Anuradha Chowdhary f, Jacques F. Meis h,i, Hamid Badali c,j,⁎ a
Department of Medical Parasitology and Mycology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran Cellular and Molecular Research Center, Urmia University of Medical Sciences, Urmia, Iran c Department of Medical Mycology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran d Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran e Université Paris-Descartes, Faculté de Médecine, APHP, Hôpital Européen Georges Pompidou, Unité de Parasitologie-Mycologie, Service de Microbiologie, Paris, France f Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India g Dynamyc Research Group (EA 7380), Paris Est Créteil University, Ecole nationale vétérinaire d'Alfort, Créteil, France h Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital (CWZ), Nijmegen, The Netherlands i Center of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands j Pharmaceutical Sciences Research Center, Hemoglobinopathy Institute, Mazandaran University of Medical Sciences, Sari, Iran b
a r t i c l e
i n f o
Article history: Received 8 September 2017 Received in revised form 19 February 2018 Accepted 3 March 2018 Available online xxxx
a b s t r a c t In vitro interaction of voriconazole with micafungin was evaluated against 33 clinical Aspergillus fumigatus isolates, including azole-resistant (n = 31) and -susceptible (n = 2) isolates. Interaction was synergistic for only 1 resistant isolate carrying the TR34/L98H mutation. No antagonistic effects were observed for 96.8% of azole-resistant isolates. © 2018 Elsevier Inc. All rights reserved.
Keywords: In vitro interactions Voriconazole Micafungin Aspergillus fumigatus
Azole-resistant Aspergillus fumigatus isolates have emerged as a major source of life-threatening aspergillosis in hospitalized patients, and these infections have been associated with significant morbidity and mortality even when properly diagnosed and treated (Lestrade et al., 2016; Verweij et al., 2016). Azole resistance, which is reported worldwide, may be due to long-term azole therapy in patients or the selective pressure of agrochemical triazoles in the environment (Chowdhary et al., 2013; Chowdhary and Meis, 2018). A vicious cycle is created as azole-resistant aspergillosis forces us to rely on limited additional available antifungal drugs. However, combination therapy may be an interesting approach to treat patients with Aspergillus infection (Johnson et al., 2004; Marr et al., 2015; Mukherjee et al., 2005; Verweij
⁎ Corresponding author. Tel.: +98-9128413720 (Mobile); fax: +98-1133543249. E-mail addresses: [email protected] (H. Fakhim), [email protected] (A. Vaezi), [email protected] (E. Dannaoui), [email protected] (C. Sharma), [email protected] (B. Mousavi), [email protected] (A. Chowdhary), [email protected] (J.F. Meis), [email protected] (H. Badali).
et al., 2016). There is some evidence supporting combination therapy with 2 antifungals with different mechanism of action (Aliff et al., 2003; Marr et al., 2015), especially the combination of echinocandins with voriconazole (Aliff et al., 2003; Cuenca-Estrella et al., 2005; Lewis and Kontoyiannis, 2005; Marr et al., 2004; Perkhofer et al., 2008). The aim of this study was to determine the in vitro interaction of voriconazole with micafungin against azole-resistant A. fumigatus clinical isolates harboring various resistance mechanisms and originating from different geographical regions. A panel of 33 well-characterized clinical A. fumigatus strains, comprising azole-resistant [i.e., TR34/L98H (n = 10), TR46/Y121F/ T289A (n = 6), G54E (n = 8), M220 (n = 3), cyp51A wild-type gene (n = 4)] and azole-susceptible (wild type; n = 2) strains, was tested. These isolates originated from the Netherlands, Romania, Tanzania, India, and Iran. All isolates were stored at −80°C at the Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, India, and also at the reference culture collection of Invasive Fungi Research Center, Mazandaran University of Medical Sciences,
https://doi.org/10.1016/j.diagmicrobio.2018.03.003 0732-8893/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Fakhim H, et al, In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus..., Diagn Microbiol Infect Dis (2018), https://doi.org/10.1016/j.diagmicrobio.2018.03.003
2
H. Fakhim et al. / Diagnostic Microbiology and Infectious Disease xxx (2018) xxx–xxx
micafungin MECs when tested alone was 0.125–N16 μg/mL and 0.002– 0.031 μg/mL, respectively. By using a complete inhibition endpoint, FICI of TR34/L98H, TR46/Y121F/T289A, G54E, M220, and isolates without cyp51A mutations ranged from 0.37 to 2.06, 0.75 to 1.5, 0.62 to 2.5, 0.5 to 1.5, and 0.62 to 1.25, respectively. The widest range of FICI-0 was found for isolates harboring the TR34/ L98H mutation. The combination exhibited indifferent activity against wild-type controls isolates. The interaction between voriconazole and micafungin was indifferent for 31 of 33 (93.9%) isolates. The combination was synergistic for only 1 isolate with TR34/L98H mutation and antagonistic for 1 azole-resistant isolate with the G54E mutation. We assessed the combination of voriconazole with micafungin against A. fumigatus harboring various azole-resistant mechanisms from different regions. In the current study, 64.5% (20 of 31) of azoleresistant strains were not susceptible to voriconazole. Although, voriconazole combined with micafungin did not exhibit a synergistic activity against 96.8% (30/31) of azole-resistant isolates, this combination against 83.9% (26/31) and 90.3% (28/31) of these isolates showed a 2- to 3-log2-dilution step lower activity than voriconazole and micafungin alone, respectively. A previous study by Seyedmousavi et al. (2013) reported a synergistic interaction of voriconazole and anidulafungin combination. However, this study also pointed out that synergistic drug interactions, based on measuring the FICI, were dependent on MIC endpoints, in which FICI was inversely related to voriconazole and anidulafungin MICs and were influenced by cyp51A genotype. Thus, voriconazole–echinocandin combination could possibly be less effective in voriconazole-resistant strains with high MICs. However, other studies
Sari, Iran. All isolates were previously identified by conventional and molecular methods, i.e., cultures supplemented with 4 μg/mL and 1 μg/mL of itraconazole and voriconazole, respectively, at 45°C for 72 h in the dark, in vitro antifungal susceptibility as described in CLSI document M38-A2 (CLSI, 2008), DNA sequencing of the partial βtubulin gene (Abastabar et al., 2016; Badali et al., 2013; Nabili et al., 2016), and sequencing of Cyp51A and promotor region (Chowdhary et al., 2015; Tamura et al., 2011). In vitro antifungal susceptibility to voriconazole (VRC; Pfizer, Groton, CT, USA) and micafungin (MFG; Astellas, Toyama, Japan) alone was determined as described in CLSI document M38-A2 (CLSI, 2008). Isolates with a voriconazole MIC of N2 μg/mL were considered resistant. The in vitro interaction between voriconazole and micafungin was tested by using a checkerboard method based on the microdilution broth reference technique (M38-A2) (CLSI, 2008; Odds, 2003). The concentration range of voriconazole (0.25 to 16 μg/mL or 0.031 to 2 μg/mL) depended on the MIC results of each isolate and was from 0.0002 to 0.125 μg/mL for micafungin. MICs/MECs were determined after 48 h of incubation at 35°C. Both complete (VisuMIC-0) and partial (VisuMIC50) inhibition endpoints were used (Arikan et al., 2002; Planche et al., 2012). The fractional inhibitory concentration index (FICI) was calculated and the interaction was interpreted as synergistic (FICI ≤ 0.5), indifferent (FICI 0.5–4), or antagonistic (FICI N 4) (Odds, 2003). Table 1 summarizes the results of in vitro interaction of voriconazole with micafungin against all A. fumigatus strains. A total of 64.5% (20 of 31) and 39.9% (13 of 33) of A. fumigatus were resistant and susceptible to voriconazole, respectively. The range of voriconazole MICs and
Table 1 In vitro interaction of voriconazole with micafungin against azole-resistant Aspergillus fumigatus isolates. Strains
Mutations
Origin
VRC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
TR46/Y121F/T289A TR46/Y121F/T289A TR46/Y121F/T289A TR46/Y121F/T289A TR46/Y121F/T289A TR46/Y121F/T289A TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H TR34/L98H G54E G54E G54E G54E G54E G54E G54E G54E M220 M220 M220 No mutation No mutation No mutation No mutation Wild type (ATCC204305) Wild type (290/E1/11)
India India Tanzania Tanzania Netherlands Netherlands Tanzania Tanzania Romania Romania India India Netherlands Netherlands Iran Iran Romania Romania Tanzania Tanzania India India Netherlands Netherlands Netherlands Netherlands Netherlands India India Iran Iran US (Virginia) India
MICs/MECs (μg/mL) in combination
MICs/MECs (μg/mL) of the drugs alone
FICI in combination
MFG
VRC
MFG
MIC
MEC
MIC-0
MIC-50
MIC-0
MEC
MIC-0
MEC
N16 16 N16 N16 N16 N16 4 8 16 16 8 4 8 8 4 16 1 1 0.125 0.125 1 0.25 0.125 0.125 0.5 0.5 8 4 16 4 4 0.125 0.125
0.008 0.016 0.004 0.002 0.016 0.031 0.016 0.008 0.016 0.016 0.008 0.016 0.008 0.016 0.016 0.016 0.008 0.004 0.016 0.016 0.008 0.016 0.016 0.008 0.016 0.008 0.016 0.016 0.016 0.016 0.016 0.004 0.008
8 4 8 16 16 16 2 8 8 8 4 1 8 16 2 8 0.25 0.5 0.125 0.063 0.25 0.125 0.125 0.063 0.125 0.25 2 2 8 0.25 4 0.125 0.063
16 1 4 8 4 8 4 8 16 16 8 4 16 16 4 16 0.25 0.25 0.063 0.063 0.125 0.125 0.25 0.125 0.125 0. 25 8 4 16 0.25 2 0.125 0.125
0.004 0.008 0.002 0.002 0.004 0.008 0.004 0.004 0.004 0.008 0.004 0.002 0.008 0.001 0.004 0.008 0.004 0.008 0.002 0.004 0.004 0.002 0.008 0.004 0.008 0.008 0.004 0.002 0.004 0.002 0.004 0.001 0.002
0.016 0.016 0.004 0.004 0.016 0.031 0.016 0.016 0.008 0.016 0.016 0.004 0.016 0.004 0.016 0.016 0.016 0.016 0.008 0.016 0.016 0.016 0.016 0.016 0.016 0.016 0.016 0.008 0.016 0.016 0.016 0.004 0.008
0.75 0.75 0.75 1.5 0.75 0.75 0.75 1.5 0.75 1 1 0.37 2 2.06 0.75 1 0.75 2.5 1.12 0.75 0.75 0.62 1.5 1 0.75 1.5 0.5 0.62 0.75 1.12 1.25 1.25 0.75
2.5 1.06 1.12 2.25 1.12 1.25 2 3 1.5 2 3 1.25 4 2.25 2 2 2.25 4.25 1 1.5 2.12 1.5 3 3 1.25 2.5 2 2 2 2 1.5 2 2
Abbreviations: TR34/L98H and TR46/Y121F/T289A = tandem repeat and mutations (promotor duplication and cyp51A substitutions); M220 and G54E = single point mutation (cyp51A substitution); no mutation = without any mutations in cyp51A gene; VRC = voriconazole; MFG = micafungin; MICs = minimum inhibitory concentration; MECs = minimum effective concentration; MIC-50 = 50% inhibition; MIC-0 = 100% inhibition; FICI = fractional inhibitory concentration index.
Please cite this article as: Fakhim H, et al, In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus..., Diagn Microbiol Infect Dis (2018), https://doi.org/10.1016/j.diagmicrobio.2018.03.003
H. Fakhim et al. / Diagnostic Microbiology and Infectious Disease xxx (2018) xxx–xxx
reported no significant differences of the combination of echinocandins with azoles against Aspergillus species (Heyn et al., 2005; Lewis and Kontoyiannis, 2005; Perkhofer et al., 2008; Planche et al., 2012; Rubin et al., 2002). Notably, our study suggested that the different resistance mechanisms of isolates originating from different geographical regions compared to wild-type control isolates cannot influence the synergistic effects of combined agents. We were unable to find a correlation between the resistant mechanisms and in the vitro combination results. The earlier reported combination of anidulafungin with voriconazole had less effect against voriconazole-resistant strains (Seyedmousavi et al., 2013). The use of different visually determined MIC endpoints, wide ranges of FIC index cutoff values, and various methodologies employed may be important factors associated with the underlying explanation for in vitro results. In our study, we used cutoff values to indicate that the interactions were synergistic if the FIC index was ≤0.5, indifferent if the FIC index was 0.5 to 4, and antagonistic if the FIC index was N4 (Odds, 2003). Evidence to support combination therapy for azole-resistant A. fumigatus infections is scarce at present. Although in vitro interactions are conducted in well-controlled conditions with accurate concentrations of multiple drugs and known inocula of spores, it can be different from the unpredictable drug concentrations and fungal burdens in patients. In addition, in vitro studies cannot take into account the effect of the host immune system. Alternative antifungal strategies including combination therapy to avoid further emergence of azole resistance at the first suspicion of invasive aspergillosis in critical ill patients can be considered as the possible resolution for the treatment of azole-resistant aspergillosis (Martín-Peña et al., 2014). In conclusion, in vitro interaction of voriconazole and micafungin was indifferent against 93.9% of azole-resistant A. fumigatus isolates. An increased likelihood that the resistant A. fumigatus will be susceptible to at least one of the components makes this a suitable option as an empiric combination regimen (Verweij et al., 2015). More in vitro and in vivo studies are necessary to address questions regarding azole and echinocandin combination therapy. Acknowledgments This study was financially supported by a grant from the Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India. Hamed Fakhim was supported by an ESCMID observership grant (ID: 1002) which is gratefully acknowledged. Hamid Badali was funded by a grant (no. 2407) from Mazandaran University of Medical Sciences, Sari, Iran. Author contributions H.F and H.B.: research concept/design, data collection, susceptibility testing, analysis, interpretation of data, and drafting article; A.V.: susceptibility testing, analysis, interpretation of data, and drafting article; E.D.: analysis, interpretation of data, critical revision of article, and approval of article; C.S.: susceptibility testing, analysis, interpretation of data; B.M.: drafting article; A.C., J.F.M. ,and H.B.: interpretation of data, and critical revision and approval of article. All authors read and approved the final manuscript. Conflict of interest J.F.M. received grants from Astellas, Merck, and Basilea. He has been a consultant to Basilea, Scynexis, and Merck and received speaker fees from Merck, Pfizer, Gilead, TEVA, and United Medical. During the past 5 years, E.D. has received research grants from MSD and Gilead; travel grants from Gilead, MSD, Pfizer, and Astellas; and speaker's fee from Gilead, MSD, and Astellas. All other authors no potential conflicts of interest.
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Please cite this article as: Fakhim H, et al, In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus..., Diagn Microbiol Infect Dis (2018), https://doi.org/10.1016/j.diagmicrobio.2018.03.003