Journal de Mycologie Médicale (2015) 25, 63—70
Available online at
ORIGINAL ARTICLE/ARTICLE ORIGINAL
Evaluation of antifungal combinations of nystatin-intralipid against Aspergillus terreus using checkerboard and disk diffusion methods ´Evaluation de combinaisons antifongiques avec la nystatine´ thodes du intralipide contre Aspergillus terreus au moyen des me damier et de la diffusion par disque R. Semis a,b, M. Nahmias a, S. Lev a, M. Frenkel a, E. Segal a,* a
Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel b City of Hope, Beckman Research Institute, Department of Immunology, Duarte, California, USA Received 21 October 2014; received in revised form 21 December 2014; accepted 31 December 2014 Available online 29 January 2015
KEYWORDS Aspergillus terreus; Nystatin-Intralipid; Antifungal combinations; Checkerboard assay; Disk-diffussion assay
Summary Objective. — The objective of this study was to evaluate the efficacy of combinations of nystatin-intralipid, found previously to be more active than nystatin, with antifungals of different mode of activity, against Aspergillus terreus. Methods. — Antifungal activity of combinations of nystatin-intralipid with voriconazole, caspofungin, terbinafine or 5-fluorocytosine were evaluated by the checkerboard and disk diffusion methods. The results were compared to those obtained with nystatin. Results. — The combination of nystatin-intralipid with caspofungin exhibited better antifungal activity than each drug alone and resulted in a synergistic interaction in three out of six tested strains of A. terreus. No such effect was obtained with Nystatin and caspofungin. Nystatinintralipid or nystatin with voriconazole yielded indifferent interactions. When nystatin-intralipid was combined with terbinafine, a strong antagonism was produced in all six A. terreus strains. This effect was observed both by checkerboard and disk diffusion methods. In contrast no interaction or only slight antagonism was observed in the combination of nystatin with terbinafine. Disk diffusion method revealed similar inhibition zones when disks impregnated
* Corresponding author. E-mail address: [email protected]
(E. Segal). http://dx.doi.org/10.1016/j.mycmed.2014.12.002 1156-5233/# 2015 Elsevier Masson SAS. All rights reserved.
R. Semis et al. with 5-fluorocytosine were placed on plain, nystatin-intralipid or nystatin containing agar plates. Conclusions. — Among four tested combinations, only combination of nytatin-intralipid with caspofungin, a representative of the echinocandin class of antifungals, resulted in synergistic interaction. Antagonism obtained by combining nystatin-intralipid with terbinafine can be explained by existence of hydrophobic interaction between these two compounds interfering with their antifungal action. The fact that nystatin-intralipid and nystatin interact differently with other antifungals, may indicate differences in their mechanisms of activity. # 2015 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Aspergillus terreus ; Nystatine-intralipide ; Combinaisons antifongiques ; Méthode du damier ; Méthode de la diffusion par disque
Re ´sume ´ Objectifs. — L’objectif de cette étude était d’évaluer l’efficacité de combinaisons de nystatineintralipide qui a été prouvée auparavant être plus active que la nystatine, avec des antifongiques ayant différents modes d’action dirigés contre Aspergillus terreus. Me ´thodes. — L’activité antifongique de combinaisons de nystatine-intralipide avec le voriconazole, caspofungin, terbinafine ou la 5-fluorocytosine a été évaluée par les méthodes du damier et de la diffusion par disque. Les résultats ont été comparés avec ceux obtenus avec la nystatine. Re ´sultats. — La combinaison de nystatine-intralipide avec la caspofungine a présenté une meilleure activité antifongique que chaque drogue testée individuellement et a resulté en une interaction synergique avec 3 des 6 souches d’A. terreus testées. Cette activité n’est pas obtenue avec la nystatine et la caspofungine seule. La nystatine-intralipide ou la nystatine avec le voriconazole ont produit des interactions indifférentes. Quand la nystatine-intralipide a été combinée avec la terbinafine, un puissant antagonisme a été obtenu avec les 6 souches d’A. terreus testées. Cet effet a été obtenu par la méthode du damier ainsi que par celle de la diffusion par disque. Par contre, aucune interaction ou éventuellement un léger antagonisme a été obtenu avec la combinaison de la nystatine et de la terbinafine. La méthode de diffusion par disque a révélé des zones similaires d’inibition quand les disques imprégnés de 5-fluorocytosine ont été placés sur des plaques d’agar sans rien ou contenant de la nystatine-intralipide ou de la nystatine. Conclusions. — Parmi les 4 combinaisons testées, seule la combinaison nystatine-intralipide avec la caspofungine, le représentant de la famille échinocandine, a produit une interaction synergique. L’antagonisme obtenu avec la combinaison de la nystatine-intralipide et la terbinafine peut s’expliquer par l’existence d’interactions hydrophobes entre les 2 substances interférant dans leur action antifongique. Le fait que la nystatine-intralipide et la nystatine interagissent différemment avec les autres antifongiques suggère des différences dans leur mécanisme d’action. # 2015 Elsevier Masson SAS. Tous droits réservés.
Introduction Systemic fungal infections in compromised patients, such as cancer patients, caused by Aspergillus and Candida species, are a significant medical problem, since these infections are characterized by severity, difficulty of management and high rate of mortality [5,8,10,30]. Although during the last decades significant progress has been made towards developing different agents for the treatment of systemic mycoses, there is still a limited number of efficacious antifungal drugs in clinical use, some of which are either associated with major adverse effects or lead to emergence of resistant strains [12,16]. Hence, enlargement of the arsenal of therapeutic possibilities for management of systemic mycoses is still required. Among infections caused by Aspergillus species, those caused by Aspergillus terreus are particularly problematic and are correlated with poor clinical outcome and higher mortality rate . As shown by Lass Floerl et al.  in a survey of 10 years, A. terreus infections were associated with a lower response rate to Amphotericin B therapy as compared with patients with infection of non-A. terreus.
In vitro, A. terreus was found to be resistant to amphotericin B. Our laboratory has developed two new formulations of polyenes: amphotericin B-intralipid (AMB-IL) and nystatinintralipid (NYT-IL). Both preparations were characterized physically and chemically, found to be stable and exhibited higher in vitro activity against pathogenic yeasts and molds than the conventional formulations [24,27,28]. Specifically, we characterized the NYT-IL preparation by assessing the particle size using ALV particle sizer (dynamic light scattering system) and determining the association of NYT with IL, immediately after preparation and after storage at different conditions . It was found that: the particle size in NYT-IL did not differ from that of IL; over 80% of NYT was in association with IL; these features did not change during storage. These measurements indicated the stability of the NYT-IL preparation. We also assessed the in vitro toxicity of the NYT-IL preparation by measuring hemolysis of sheep red blood cells
Antifungal combinations against Aspergillus terreus (SRBC) and leakage of potassium. These assays revealed that the NYT-IL formulation is less toxic than NYT. Antifungal activity of NYT-IL was tested in vitro against 35 strains of Candida, Aspergillus and Fusarium species and in most cases revealed lower MIC values than the conventional NYT formulation, indicating improved activity . Specifically, 17 strains of five Candida species (Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis) and 12 strains of four Aspergillus species (Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger) were tested. All Candida and Aspergillus species had lower MIC values than those of NYT. In Candida species the mean MIC values for NYT-IL were <1 mg/ mL—1.4 mg/mL vs. 1.4 mg/mL— 2.2 mg/mL for NYT. The P values of the mean MIC values for each species of Candida in comparing NYT-IL vs. NYT were <0.01—0.02. Aspergillus species mean MIC values for NYT-IL ranged from <1 mg/ mL—> 3 mg/mL vs. 2 mg/mL—> 4.5 ug/mL for NYT, with P values ranging from <0.01—0.05. The better activity of NYT-IL could possibly be explained, as shown by EM analyses, by a more severe disruption of the fungal cells. AMB-IL is non-toxic in vivo and can be administered at higher concentrations than conventional AMB, thus exhibiting better efficacy in experimental C. albicans and A. fumigatus infections [25,26,28]. NYT-IL, contrary to the conventional nystatin (NYT), which is limited to topical use due to poor water solubility and toxicity upon parenteral administration [7,14], can be administered parenterally, as evaluated in an experimental animal model [22,28]. Thus, while use of NYT is limited to therapy of dermal and mucosal infections, NYT-IL can be considered as a potential antifungal drug for therapy of invasive mycoses. NYT-IL was found to be well tolerated in experimental animals and active against an experimental C. albicans systemic infection in naïve and immunocompromised mice . A study in an experimental A. fumigatus infection revealed as well antifungal activity . Use of combinations of available antifungal agents with different mechanisms of activity could be considered a rational approach to increase the efficacy of therapeutic possibilities. This was shown in vitro and in experimental animal studies, and even in clinical studies . Reed et al.  reported on the use of a combination consisting of a lipid formulation of amphotericin B (ABLC) with caspofungin for treatment of patients with rhino-cerebral mucormycosis. Moreover, some combinations are established therapeutic regimens in clinical use, such as the use of the combination of amphotericin B and 5-flurocytosine for treatment of cryptococcal meningoencephalitis . We have reported previously on the in vitro antifungal activity against C. albicans of the experimental drug nikkomycin Z in combination with caspofungin, voriconazole or amphotericin B . Furthermore, Sionov et al.  have demonstrated in an animal model of A. fumigatus infection better therapeutic activity of amphotericin B-intralipid in combination with caspofungin. Following this rational, the present study concentrated on the exploration of the in vitro activity of antifungal combinations against A. terreus of the intralipid formulation of nystatin, namely, nystatin-intralipid with representative drugs of the major groups of antifungals currently in use
65 in therapy of mycoses: triazoles, echinocandins, allylamines, and antimetabolites in comparison to combinations with the conventional formulation of nystatin. Specifically, the combinations tested included: NYT-IL or NYT and voriconazole, NYT-IL or NYT and caspofungin, NYT-IL or NYT and terbinafine, NYT-IL or NYT and 5-fluorocytosine, using two evaluation methods: the checkerboard and disk diffusion.
Materials and methods Inoculum Six A. terreus clinical strains, identified by standard accepted methods of macroscopic and microscopic morphological characteristics , were used in this study: A6, A11, N7, AT1, AT2, Y3. The cultures were grown on potato dextrose agar (PDA) (Difco) slants at 37 8C for 4—6 days prior to the experiment. Conidial suspensions were prepared according to CLSI M38-A2 protocol for molds  and adjusted to the required concentration by counting in a hematocytometer. The inoculum’s size was rechecked by counting colony-forming units (CFU) on Sabouraud’s dextrose agar (SDA) (Difco) plates.
Antifungal agents The antifungal agents are: nystatin (NYT) (Sigma Chemical, USA) stock solution 25 mg/mL was prepared by dissolving in dimethyl sulfoxide (DMSO) (Sigma Chemicals, St. Louis, Mo., USA); NYT-IL was prepared as described previously  by a 25fold dilution of NYT in IntralipidTM 20% (Kabi Pharmacia, Stockholm, Sweden) to a final NYT concentration of 1.0 mg/ml, and then agitated vigorously at 24 8C for 18 h on a controlled environmental incubator shaker (New Brunswick Scientific Co., NJ, USA) at 280 rpm; terbinafine (Kayman Chemicals) and 5-fluorocytosine (5FC) (Sigma-Aldrich) powders were dissolved in DMSO or DDW, respectively, to obtain stock solutions at concentration of 3.2 mg/mL; commercially available voriconazole (Vfend, Pfizer) was reconstituted with DDW according to manufacturer’s instructions to concentration of 5 mg/mL. caspofungin (Merck & Co) stock solutions were subsequently diluted to a working concentrations with RPMI 1640 medium (Sigma) buffered with MOPS (Applichem GmbH).
Antifungal Assessment Antifungal assessment was performed according to the microbroth dilution method of CLSI for filamentous fungi , based on determination of minimal inhibitory concentration (MIC) or minimal effective concentration (MEC) (for tests with caspofungin).
Drug combinations Activity of drug combinations was assessed using the checkerboard method and/or disk diffusion method.
R. Semis et al.
Checkerboard method The checkerboard method is an established technique to determine the activity of drug combinations . It determines the MIC of each drug in the combination and the activity of the combination. As described below, by using a formula which takes into account the mutual effects of each drug on the other component it is possible to determine whether there is a synergistic or antagonistic effect by using the two drugs together, or no effect at all as compared to the single drug. Technically, a two-fold concentration gradient of each couple of drugs was generated in 96-wells microtiter plates (Biosigma, Italy) in a two-dimensional manner. Resulted final concentration range was 1—64 mL/mL for NYT-IL and NYT, and 0.015—16 mL/mL for voriconazole, terbinafine and caspofungin. One hundred ml of fungal suspension at concentration of 1—5 104 spores/mL were added to each well. The total final volume in each well was 200 mL. The plates were incubated at 37 8C for 48 h. The MICs were determined as the lowest drug concentration with no visible growth. The minimal effective concentration (MEC) of caspofungin was determined as the lowest concentration causing abnormal hyphal growth, as detected by an inverted microscope. Drug interactions were assessed in terms of fractional inhibitory concentration index (FICI), which was calculated as follows: MICA in combination/MICA alone + MICB in combination/MICB alone (MICA and MICB indicate the MICs of drug A and B, respectively, MECs were used for caspofungin). Drug
interactions were classified as synergistic (FICI 0.5), no interaction/indifference (0.5 < FICI < 4), or antagonistic (FICI 4). Disk diffusion method Disk diffusion method is an additional recognized technique to assess in vitro susceptibility of microorganisms to various drugs . Specifically, yeast nitrogen broth (YNB) (Difco) agar plates containing 0, 2, 4 or 8 mg/mL NYT-IL or NYT were inoculated with a 106 spores suspension by swabbing the agar surface. After the plates were allowed to dry, sterile paper disks containing 1 mg/mL 5-FC (Bio-Rad) or terbinafine (homemade) were placed on the agar surface. The inhibition zones were measured after incubation at 37 8C for 48 h for plates with terbinafine or 24 h for plates with 5-FC.
Results The data for all 8 combinations of NYT-IL and NYT assessed by the checkerboard assay are summarized in Tables 1—3 and those evaluated by the disk diffusion method are presented in Figs. 1 and 2.
Combination of NYT-IL with caspofungin The results of combination of NYT-IL or NYT with caspofungin are shown in Table 1. Combination of NYT-IL with caspofungin
Table 1 Antifungal combinations with caspofungin. Combinaisons antifongiques avec la capsofungine. Strain Drug combination
N7 A6 A11 AT1 AT2 Y3
NYT + Caspo NYT-IL + Caspo NYT + Caspo NYT-IL + Caspo NYT + Caspo NYT-IL + Caspo NYT + Caspo NYT-IL + Caspo NYT + Caspo NYT-IL + Caspo NYT + Caspo NYT-IL + Caspo
MEC range (median) (mg/L) Alone
NYT Caspofungin preparation
4—8 4—8 4—4 4—4 4—8 4—8 4—8 4—4 4—8 4—4 8—8 4—4
4—8 (4) 0.25—0.5 (0.38) 1.5—5 (2) 0.5—8 (4) 0.03—0.25 (0.13) 0.38—2 (1.5) 4—8 (4) 0.25—0.5 (0.38) 2—5 (2.5) 0.5—4 (2.25) 0.01—0.5 (0.16) 0.19—2 (0.94) 4—8 (6) 0.25—0.5 (0.5) 1.5—9 (2.5) 0.5—8 (4) 0.02—0.25 (0.19) 0.63—2 (1.38) 8—8 (8) 0.13—0.25 (0.25) 3—4 (3.5) 1—2 (1) 0.004—0.03 (0.02) 0.38—0.63 (0.38) 4—8 (8) 0.13—0.25 (0.25) 2—5 (3) 0.5—1 (0.5) 0.01—0.13 (0.02) 0.16—0.63 (0.28) 8—8 (8) 0.13—0.5 (0.2) 2—2 (2) 0.5—2 (1) 0.02—0.13 (0.03) 0.2—1 (0.38)
(4) (4) (4) (4) (6) (4) (6) (4) (4) (4) (8) (4)
0.06—1 0.06—0.5 0.13—0.5 0.13—1 0.03—0.5 0.03—1 0.06—0.13 0.03—0.25 0.06—0.25 0.25—0.25 0.13—0.5 0.13—0.4
(0.38) (0.19) (0.19) (0.25) (0.38) (0.31) (0.13) (0.13) (0.19) (0.25) (0.2) (0.25)
FICI range (median)
Type No interaction No interaction No interaction No interaction No interaction No interaction No interaction Synergy No interaction Synergy No interaction Synergy
MEC: minimal effective concentration. MEC is used for assessing susceptibility to caspofungin; NYT: nystatin; NYT-IL: nystatin-intralipid; Caspo: caspofungin. FICI: fractional inhibitory concentration index which was calculated as follows: MICA in combination/MICA alone + MICB in combination/MICB alone. Drug interactions were classified as synergistic (FICI 0.5), no interaction (0.5 < FICI < 4), or antagonistic (FICI 4). Data from 3 experiments.
Antifungal combinations against Aspergillus terreus
Table 2 Antifungal combinations with voriconazole. Combinaisons antifongiques avec le voriconazole. Strain
MIC range (median) (mg/L) In combination
N7 A6 A11 AT1 AT2 Y3
NYT + Vori NYT-IL + Vori NYT + Vori NYT-IL + Vori NYT + Vori NYT-IL + Vori NYT + Vori NYT-IL + Vori NYT + Vori NYT-IL + Vori NYT + Vori NYT-IL + Vori
FICI range (median)
4—8 (4) 4—8 (4) 4—8 (8) 4—8 (8) 8—8 (8) 4—8 (4) 4—8 (4) 4—4 (4) 4—8 (8) 4—8 (4) 8—8 (8) 4—8(4)
0.13—0.25 0.13—0.25 1—1 0.25—1 0.13—0.25 0.25—0.25 0.25—0.25 0.25—0.25 0.25—0.25 0.25—0.25 0.5—0.5 0.5—0.5
8—16 8—8 2—16 8—16 4—8 8—8 4—8 4—8 8—16 8—16 4—8 2—4
0.13—0.13 0.13—0.13 0.25—1 0.25—1 0.13—0.13 0.13—0.25 0.13—0.25 0.13—0.25 0.13—0.25 0.25—0.25 0.25—0.5 0.25—0.5
2.5—3 1.5—3 0.75—3 2—3 1—2 2—3 1.5—3 1.5—3 1.5—3 3—5 1—2 1—2
No No No No No No No No No No No No
(0.25) (0.25) (1) (0.38) (0.19) (0.25) (0.25) (0.25) (0.25) (0.25) (0.5) (0.5)
(8) (8) (16) (8) (8) (8) (8) (8) (8) (16) (8) (4)
(0.13) (0.13) (0.75) (0.25) (0.13) (0.25) (0.25) (0.13) (0.25) (0.25) (0.5) (0.25)
(2.5) (2.5) (2.75) (2.25) (1.75) (2.5) (2) (2.5) (3) (3) (2) (1)
interaction interaction interaction interaction interaction interaction interaction interaction interaction interaction interaction interaction
MIC: minimal inhibitory concentration; NYT: nystatin; NYT-IL: nystatin-intralipid; Vori: voriconazole. FICI: fractional inhibitory concentration index which was calculated as follows: MICA in combination/MICA alone + MICB in combination/MICB alone. Drug interactions were classified as synergistic (FICI 0.5), no interaction (0.5 < FICI < 4), or antagonistic (FICI 4). Data from 3 experiments.
Table 3 Antifungal combinations with terbinafine. Combinaisons antifongiques avec la terbinafine. Strain
MIC range (median) (mg/L) Alone
N7 A6 A11 AT1 AT2 Y3
NYT + Terb NYT-IL + Terb NYT + Terb NYT-IL + Terb NYT + Terb NYT-IL + Terb NYT + Terb NYT-IL + Terb NYT + Terb NYT-IL + Terb NYT + Terb NYT-IL + Terb
FICI range (median)
8—8 4—4 2—4 4—4 4—8 4—4 8—8 4—8 4—8 4—8 8—8 4—8
0.13—0.5 0.06—0.5 0.13—0.5 0.13—0.5 0.25—0.5 0.06—0.5 0.06—0.13 0.06—0.13 0.06—0.06 0.06—0.06 0.06—0.13 0.06—0.13
8—16 8—16 8—16 16—16 2—16 8—16 8—16 4—16 8—8 8—8 8—8 8—16
0.13—0.13 2—8 0.06—0.25 4—8 0.13—0.25 2—8 0.06—0.13 2—4 0.06—0.06 2—4 0.06—0.13 2—4
2—2.25 (2.25) 18—34 (20) 4.25—4.5 (4.5) 12—36 (20) 0.75—4.5(2.63) 18—34 (18) 2—3 (3) 18—34 (33) 2—3 (2) 33—65 (34) 2—2 (2) 33—66 (34)
No interaction Antagonism Antagonism Antagonism No interaction Antagonism No interaction Antagonism No interaction Antagonism No interaction Antagonism
(8) (4) (4) (4) (8) (4) (8) (4) (8) (8) (8) (8)
(0.5) (0.25) (0.5) (0.5) (0.38) (0.19) (0.06) (0.13) (0.06) (0.06) (0.06) (0.06)
(16) (8) (16) (16) (16) (8) (8) (8) (8) (8) (8) (8)
(0.13) (4) (0.13) (4) (0.19) (3) (0.13) (2) (0.06) (2) (0.06) (4)
MIC: minimal inhibitory concentration; Terb: terbinafine; NYT: nystatin; NYT-IL: nystatin-intralipid. FICI: fractional inhibitory concentration index which was calculated as follows: MICA in combination/MICA alone + MICB in combination/MICB alone. Drug interactions were classified as synergistic (FICI 0.5), no interaction (0.5 < FICI < 4), or antagonistic (FICI 4). Data from 3 experiments.
R. Semis et al.
Figure 1 Antifungal combinations by disk diffusion method. Terb: terbinafine; NYT: nystatin; NYT-IL: nystatin-intralipid; 5-FC: 5-fluorocytosine. ´ thode de diffusion par disque. Combinaisons antifongiques par la me
resulted in an improvement of antifungal activity of each drug. Furthermore, synergistic interaction was observed in three out of six tested A. terreus strains. In contrast, the interaction between NYT and caspofungin was indifferent in all six tested strains.
Combination of NYT-IL with voriconazole Table 2 demonstrates data regarding combination of NYT preparations, NYT-IL or NYT, with voriconazole. The combination of each pair of drugs did not reveal improvement over the activity against A. terreus of each drug alone. Thus, no synergism or antagonistic effect was noted.
Combination of NYT-IL with terbinafine The assessment of the interaction between nystatin-intralipid and terbinafine revealed strong antagonism (Table 3). When the antifungal activity of the combination of NYT-IL and terbinafine was re-evaluated by the disk diffusion method, the inhibition zone around terbinafine-containing disk dramatically decreased with the addition of NYT-IL
into the agar (Fig. 1B), thus, confirming the antagonistic effect noted by the checkerboard technique. In contrast, assessment of the antifungal effect of a combination of NYT with terbinafine revealed no interaction in five A. terreus strains and only slight antagonism in one A. terreus strain. Fig. 1A shows fungal growth on the control plates containing no drug or only one drug, NYTIL or NYT, added into the agar.
Combination of NYT-IL with 5-FC Since the MIC values of 5-FC against A. terreus were inappropriately high for the checkerboard method, the combinations of NYT-IL or NYT with 5FC were tested by the disk diffusion method only, and are presented in Fig. 1C. As shown in Fig. 1C, the edges of the inhibition zone around the 5-FC containing disk are blurred, which might reflect the fungistatic nature of the antifungal effect of the drug. When NYT-IL was added into the agar it did not change noticeably the size of the inhibition zone. A similar effect was observed on NYT containing plates. Hence, the results indicate a state of indifference.
Antifungal combinations against Aspergillus terreus
Figure 2 Antifungal effect of intralipid with terbinafine. IL: intralipid; Terb: terbinafine. Action antifongique de l’intralipide avec la terbinafine.
Discussion A. terreus is a rare but clinically important pathogen. Infections caused by A. terreus are difficult to treat partially due to inherent resistance of A. terreus to polyene antifungal drugs. However, Intralipid formulation of nystatin, developed in our laboratory, showed better activity against A. terreus in comparison to NYT, though still not optimal. It is important to note that incorporation of another polyene, amphotericin B, into intralipid emulsion increased its activity against A. terreus, as well (data not shown). To further improve antifungal effect of NYT-IL against A. terreus, NYT-IL was combined with other antifungal drugs: caspofungin, voriconazole, terbinafine and 5FC. The only combination, which produced a synergistic effect is that of NYT-IL with caspofungin. Caspofungin
69 belongs to the echinocandin class of antifungals with a different mode of action: echinocandins act on the fungal cell wall, whereas polyenes impair membrane function. Previously, we reported significantly improved efficacy achieved by combination of AMB or AMB-IL with caspofungin against A. fumigatus in vitro and in vivo . There are several other studies that also observed synergy between polyenes and echinocandins against various pathogenic molds in vitro and in clinical studies [1,3]. Since a synergistic interaction was observed only with NYT-IL but not with NYT, it may be concluded that NYT-IL and NYT exhibit different patterns of activity. Combination of NYT-IL with voriconazole resulted in the unchanged antifungal activity of each drug (indifferent interaction). Other investigators that reported indifference or even antagonism between AMB and azoles, suggested several explanations of this evidence. Among them Sugar  proposed that as a result of ergosterol depletion from the membrane by azoles through inhibition of the ergosterol syntesis, AMB is deprived of its target molecule and opportunity to achieve its full potency. Scheven and Schwegler  suggest that lipophilic azoles can be absorbed in the fungal cell membrane directly impeding AMB’s binding with ergosterol. Interaction of NYT with terbinafine was for the most part indifferent or slightly antagonistic. Surprisingly, combination of NYT-IL with terbinafine resulted in strong antagonism. This phenomenon recurred in both methods used to examine susceptibility of A. terreus to antifungal combinations: checkerboard and disk diffusion methods. When terbinafine was combined with intralipid alone, decrease in antifungal activity of terbinafine was observed to a similar extent (Fig. 2). The most likely conclusion from these findings is that terbinafine, being a hydrophobic compound , interacts with the intralipid moiety of NYT-IL, to reduce antifungal potency of the antifungal combination. As indicated in the ‘‘Results’’ the combinations of NYT-IL and NYT with 5FC were tested by disk diffusion method only. 5-FC impregnated disks produced similar inhibition zones on the surface of solid medium with or without NYT-IL. Likewise, comparable inhibition zones around the 5-FC disks were observed on NYT containing plates. These findings suggest indifferent interaction between 5FC and NYT-IL as well as between 5FC and NYT against A. terreus. Another recent study revealed that, although, disturbance of nucleotide metabolic pathways by 5-FC or by other means potentiates activity of AMB against Cryptoccocus and Candida, it does not affect AMB activity against Aspergillus, explained by differences in nucleotide requirements and metabolic machinery between these fungal organisms . This study showed that the two tested NYT formulations exhibit different types of interaction with other antifungal drugs such as caspofungin and terbinafine. This evidence may be a consequence of a different mechanism of action of these two NYT preparations, which was already noted in our previous studies exploring mechanism of action of NYT-IL vs. NYT . Furthermore, in vivo experiments demonstrated that NYT-IL and NYT exhibit different tissue distribution pattern, immunomodulatory effect, toxicity and, finally, the overall therapeutic effect, in favor of NYT-IL [22,23]. Obviously, modifications in drug formulation may have a significant impact on various parameters and effects exerted
70 by the drug, probably, through alterations in multi-level interactions between the drug and diverse components of complex biological systems of a fungal pathogen and, even more so, of a host organism. The combination of NYT-IL with caspofungin appears to be promising against A. terreus meriting further in vivo investigations. In addition, NYT-IL/caspofungin combination can be tested against other fungal pathogens, particularly those known to be resistant to standard antifungal treatments.
Disclosure of interest Semis Rita, Nahmias Moshe, Lev Sigalit were graduate students at the Sackler School of Medicine at Tel-Aviv University and the funds for the research were University funds and no outside sources. Thus, there is no conflict of interest involved.
References  Arikan S, Lozano-Chiu M, Paetznick V, Rex JH. In vitro synergy of caspofungin and amphotericin B against Aspergillus and Fusarium spp. Antimicrob Agents Chemother 2002;46:245—7.  Banerjee D, Burkard L, Panepinto JC. Inhibition of nucleotide biosynthesis potentiates the antifungal activity of amphotericin B. PLoS One 2014;9:e87246.  Caillot D, Thieberbrecht R, Herbrecht R, de Botton S, Pigneux A, Bernard F, et al. Liposomal amphotericin B in combination with caspofungin for invasive aspergillosis in patients with hematologic malignancies: a randomized pilot study (Combistrat trial). Cancer 2007;110:2740—6.  Clinical, Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, Second Edition: Approved Standard M38-A2. Wayne, PA, USA: CLSI; 2008.  de Pauw B. Antifungal therapy. Transplant Proc 2011;43: 2461—2.  Gianni C. Update on antifungal therapy with terbinafine. G Ital Dermatol Venereol 2010;145:415—24.  Hazen EL, Brown R. Nystatin. Ann N Y Acad Sci 1960;89:258—66.  Hope W, Natarajan P, Goodwin L. Invasive fungal infections. Clin Med 2013;13:507—10.  Johnson EM, Espinel-Ingroff AV, Pfaller MA. Susceptibility test methods: yeasts and filamentous fungi. In: Versalovic J, et al., editors. 10th ed., Manual of Clinical Microbiology, vol. 2, 10th ed. Washington DC: ASM; 2011. p. 2020—37.  Kurosawa M, Yonezumi M, Hashino S, Tanaka J, Nishio M, Kaneda M, et al. Epidemiology and treatment outcome of invasive fungal infections in patients with hematological malignancies. Int J Hematol 2012;96:748—57.  Lass-Florl C, Griff K, Mayr A, Petzer A, Gastl G, Bonatti H, et al. Epidemiology and outcome of infections due to Aspergillus terreus: 10-year single centre experience. Br J Haematol 2005;131:201—7.  Lewis RE. Current concepts in antifungal pharmacology. Mayo Clin Proc 2011;86:805—17.  Mukherjee PK, Sheehan DJ, Hitchcock CA, Ghannoum1 MA. Combination treatment of invasive fungal infections. Clin Microbiol Rev 2005;18:163—94.
R. Semis et al.  Ng AW, Wasan KM, Lopez-Berestein G. Development of liposomal polyene antibiotics: an historical perspective. J Pharm Pharm Sci 2003;6:67—83.  Perfect JR, DismukesWE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of America. Clin Infect Dis 2010;50:291—322.  Pitman SK, Drew RH, Perfect JR. Addressing current medical needs in invasive fungal infection prevention and treatment with new antifungal agents, strategies and formulations. Expert Opin Emerg Drugs 2011;16:559—86.  Reed C, Bryant R, Ibrahim AS, Edwards J, Filler SG, Goldberg R, et al. Combination polyene-caspofungin treatment of rhinoorbital-cerebral mucormycosis. Clin Infect Dis 2008;47:364—71.  Larone DH. The genus Aspergillus, in medically important fungi, a guide to identifican, . 5th ed., Washington D.C, USA: ASM Press; 2011: 281—93.  Sandovsky-Losica H, Shwartzman R, Lahat Y, Segal E. Antifungal activity against Candida albicans of nikkomycin Z in combination with caspofungin, voriconazole or amphotericin B. J Antimicrob Chemother 2008;62:635—7.  Scheven M, Schwegler F. Antagonistic interactions between azoles and amphotericin B with yeasts depend on azole lipophilia for special test conditions in vitro. Antimicrob Agents Chemother 1995;39:1779—83.  Semis R, Kagan S, Berdicevsky I, Polacheck I, Segal E. Mechanism of activity and toxicity of nystatin-intralipid. Med Mycol 2013;51:422—31.  Semis R, Mendlovic S, Polacheck I, Segal E. Activity of an Intralipid formulation of nystatin in murine systemic candidiasis. Int J Antimicrob Agents 2011;38:336—40.  Semis R, Nili SS, Munitz A, Zaslavsky Z, Polacheck I, Segal E. Pharmacokinetics, tissue distribution and immunomodulatory effect of intralipid formulation of nystatin in mice. J Antimicrob Chemother 2012;67:1716—21.  Semis R, Polacheck I, Segal E. Nystatin-intralipid preparation: characterization and in vitro activity against yeasts and molds. Mycopathologia 2010;169:333—41.  Shadkchan Y, Segal E. Antifungal activity of amphotericin B-lipid admixtures in experimental systemic candidosis in naive mice. J Antimicrob Chemother 1999;44:787—90.  Shadkchan Y, Segal E. Treatment of experimental candidosis with amphotericin B-intralipid admixtures in immunocompromised mice. J Antimicrob Chemother 2001;48:245—51.  Shadkhan Y, Segal E, Bor A, Gov Y, Rubin M, Lichtenberg D. The use of commercially available lipid emulsions for the preparation of amphotericin B-lipid admixtures. J Antimicrob Chemother 1997;39:655—8.  Sionov E, Segal E. Treatment of murine systemic aspergillosis with polyene-intralipid admixtures. Med Mycol 2004;42:73—80.  Sionov E, Mendlovic S, Segal E. Efficacy of amphotericin B or amphotericin B-intralipid in combination with caspofungin against experimental aspergillosis. J Infect 2006;53:131—9.  Sipsas NV, Kontoyiannis DP. Invasive fungal infections in patients with cancer in the Intensive Care Unit. Int J Antimicrob Agents 2012;39:464—71.  Steinbach WJ, Benjamin DK, Kontoyiannis DP, Perfect JR, Lutsar I, Marr KA, et al. Infections due to Aspergillus terreus: a multicenter retrospective analysis of 83 cases. Clin Infect Dis 2004;39:192—8.  Sugar AM. Antifungal combination therapy: where we stand. Drug Resist Updat 1998;1:89—92.