Efficacy of Antifungal Monotherapies and Combinations against Aspergillus calidoustus

Invasive fungal infections due to Aspergillus calidoustus with decreased azole susceptibility are emerging in the setting of azole prophylaxis and are associated with poor outcomes. We assessed the in vitro activity of antifungal drugs used alone or in combinations against A. calidoustus and found a synergistic effect between voriconazole and terbinafine at concentrations within the therapeutic range.

ABSTRACT

Invasive fungal infections due to Aspergillus calidoustus with decreased azole susceptibility are emerging in the setting of azole prophylaxis and are associated with poor outcomes. We assessed the in vitro activity of antifungal drugs used alone or in combinations against A. calidoustus and found a synergistic effect between voriconazole and terbinafine at concentrations within the therapeutic range. An invertebrate Galleria mellonella model of A. calidoustus infection tended to support the potential benefit of this combination.

INTRODUCTION

Invasive aspergillosis is a severe infection affecting immunocompromised hosts. While Aspergillus fumigatus remains the predominant pathogenic species, Aspergillus calidoustus (section Usti) is an emerging pathogen exhibiting some degree of intrinsic azole resistance and causing breakthrough infections in patients receiving antimold prophylaxis (1–3). Dissemination to soft tissues or the brain is frequently observed in A. calidoustus infections, which are often refractory to antifungal therapy and associated with high mortality rates (3). As a result, many patients are treated with combinations of antifungals expecting some synergistic interactions, which has not been demonstrated. The aim of this study was to assess the in vitro and in vivo efficacy of antifungal monotherapies and combinations against A. calidoustus.

(Preliminary results of this research were presented as a poster at the 28th European Congress of Clinical Microbiology and Infectious Diseases [ECCMID], Madrid, Spain, 21 to 24 April 2018 [4]).

Ten clinical isolates of A. calidoustus were selected for this study. Species identification was confirmed by partial sequencing of the beta-tubulin (BenA) and calmodulin (CaM) genes, as previously described (5). Drugs were obtained as powders (Sigma-Aldrich, St. Louis, MO), dissolved in dimethyl sulfoxide (DMSO) for a stock concentration of 5 mg/ml, and stored at −20°C. In vitro antifungal susceptibility testing was performed according to the M38-A3 Clinical and Laboratory Standards Institute (CLSI) broth microdilution method (6). Plates were incubated at 35°C and read by visual inspection at 48 h. Synergy testing was performed by the checkerboard dilution method as previously described, with interactions defined as synergistic, indifferent, or antagonistic for a fractional inhibitory concentration index (FICI) of ≤0.5, >0.5 to 4, and >4, respectively (7). Amphotericin B, voriconazole, posaconazole, isavuconazole, and terbinafine were tested alone and in combinations. Experiments were performed in duplicates. In the case of discordant results (FICI difference of >0.05), a third replicate was performed, and the result was expressed as the FICI for which two concordant results were obtained.

An invertebrate model of A. calidoustus infection was performed in Galleria mellonella larvae (Bait Express GmbH, Basel, Switzerland) as previously described (8). Groups of 20 larvae with a weight ranging from 350 to 400 mg were infected with a single dose of 3 × 10⁶ spores in 40 µl phosphate-buffered saline (PBS). Antifungal drugs were injected 2 h later as a single 40-µl dose of amphotericin B (5 mg/kg), voriconazole (10 mg/kg), terbinafine (5 mg/kg), or a combination of voriconazole (10 mg/kg) and terbinafine (5 mg/kg). Drug doses were selected based on previous pharmacokinetic models in G. mellonella or extrapolated from human dosage (9, 10). Larvae were incubated at 37°C, and survival was assessed twice daily during 7 days postinfection. Performing a single injection of antifungal treatment 2-h postinfection, we analyzed the survival curve by the Gehan-Breslow-Wilcoxon test with GraphPad Prism software, giving more power to deaths at early time points.

MICs of amphotericin B, voriconazole, posaconazole, isavuconazole, and terbinafine are shown in Table 1. The combination of voriconazole and terbinafine was tested on all strains, with a synergistic effect (FICI, ≤0.5) in 9/10 strains (Table 1). Other drug combinations were tested on strains 1 and 2, with the following FICI results: posaconazole-terbinafine, 0.5 (synergistic); isavuconazole-terbinafine, 0.4 and 0.6 (synergistic and indifferent), amphotericin B-voriconazole, 3 (indifferent); and amphotericin B-terbinafine, >4 (antagonistic). Caspofungin, exhibiting only very modest fungistatic activity against A. calidoustus, showed indifferent interactions with either voriconazole or amphotericin B (FICI, 2).

TABLE 1.

In vitro activity of antifungal drugs alone and combined against 10 Aspergillus calidoustus strains

Strain	MIC (µg/ml) for antifungal drugsa:					MICs (µg/ml) for VOR-TBFb	FICI for VOR-TBFb
	AMB	VOR	POS	ISA	TBF
1	1	4	8	2	0.5	1, 0.12	0.5
2	1	4	8	2	1	1, 0.25	0.5
3	1	8	>16	2	0.5	1, 0.25	0.6
4	0.5	4	>16	4	1	1, 0.25	0.5
5	0.5	8	>16	4	1	2, 0.25	0.5
6	0.25	8	>16	4	0.5	2, 0.12	0.5
7	1	2	8	2	0.5	0.5, 0.12	0.5
8	1	2	8	2	1	0.25, 0.25	0.4
9	2	8	4	2	1	2, 0.25	0.5
10	0.5	8	>16	4	0.5	2, 0.12	0.5

^aAMB, amphotericin B; VOR, voriconazole; POS, posaconazole; ISA, isavuconazole; TBF, terbinafine.

^bMIC of voriconazole and terbinafine when used in combination. FICI, fractional inhibitory concentration index.

To further assess the efficacy of antifungal drugs against A. calidoustus, we tested the different monotherapies and the voriconazole-terbinafine combination in a G. mellonella infection model using A. calidoustus strain 1. The experiment was performed in duplicates (Fig. 1A and B). Each individual drug (amphotericin B, voriconazole, and terbinafine) demonstrated a significant effect compared with the untreated group, but there was no superiority of one drug compared with another. The voriconazole-terbinafine combination was associated with a markedly improved survival compared with the untreated group in both experiments (P < 0.0001). Compared with monotherapies, the superiority of the combination therapy reached statistically significant P values against terbinafine alone in the first experiment and against both voriconazole and amphotericin B in the second experiment.

FIG 1.

Efficacy of antifungal drugs in a Galleria mellonella model of A. calidoustus infection. Survival curves of G. mellonella larvae injected with 3 × 10⁶ spores of A. calidoustus and a single dose of antifungal drug 2-h postinfection. Larvae were monitored for survival twice daily. Groups of 20 larvae were used each time. (A and B) The results of two independent experiments. (C) Data were analyzed by Gehan-Breslow-Wilcoxon test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. PBS, phosphate-buffered saline (no drug); AMB, amphotericin B; VOR, voriconazole; TBF, terbinafine.

This in vitro and in vivo analysis of the activity of antifungal drugs used alone or in combination against Aspergillus calidoustus lead to some interesting conclusions. First, despite better in vitro activity, amphotericin B did not demonstrate superiority over voriconazole in an invertebrate model of infection. Indeed, voriconazole demonstrated a significant in vivo efficacy against this fungus despite MIC values that are usually considered at the limit or above the therapeutic range of concentrations. The addition of terbinafine to voriconazole or other mold-active azoles (posaconazole and isavuconazole) resulted in a moderate positive interaction at the limit of the synergistic criteria (FICI approximately 0.5). Low concentrations of terbinafine (0.12 to 0.25 µg/ml) were sufficient to lower the voriconazole MIC from 4 to 8 µg/ml to 1 to 2 µg/ml, which corresponds to targeted therapeutic serum concentrations for the treatment of invasive aspergillosis (11, 12). The invertebrate model of A. calidoustus infection tended to support the potential benefit of the voriconazole-terbinafine combination despite some discrepant results between the two experiments. Such variability of the fitness of the Galleria spp. larvae is frequently observed (8), which may be due to their heterogeneous genetic background, age and stage of seasonal cycle, or mode of preservation. Despite variability, the voriconazole-terbinafine combination was associated with the highest survival rate compared with the untreated group in both experiments (P < 0.0001) and was the only treatment arm showing some significant improvement compared with the others. The second experiment, associated with a higher early mortality rate, may be more representative to assess significant differences between treatment arms. In this last experiment, the voriconazole-terbinafine combination was significantly more effective compared with both voriconazole or amphotericin B alone. Albeit not tested in the Galleria mellonella model, other drug combinations, such as amphotericin B associated with voriconazole or terbinafine, resulted in indifferent or even deleterious interactions in vitro.

Topical or systemic terbinafine has been anecdotally used as adjunctive antifungal treatment for primary cutaneous infections due to A. calidoustus in the past (13, 14). Terbinafine is known for its accumulation in soft tissues and can also penetrate the hematoencephalic barrier (15); thus, it represents an interesting adjuvant therapy for A. calidoustus infections that frequently affect the soft tissues and brain (29% and 12% of cases, respectively) (3). The combination of voriconazole and terbinafine has already demonstrated some synergism against other Aspergillus spp. or other molds (e.g., Scedosporium spp.) and has been used for the treatment of invasive scedosporiosis (16–18).

A. calidoustus infection remains a rare but often fatal disease, for which the optimal antifungal treatment is still debated. Because of the intrinsic level of azole resistance of this fungus, amphotericin B is usually the preferred first-line antifungal therapy, but its use is limited by nephrotoxicity and the lack of an oral formulation for prolonged therapy. Although this study was performed with a limited number of isolates, our results showed that, despite better in vitro antifungal activity, amphotericin B did not demonstrate better efficacy compared with voriconazole in a Galleria mellonella model of infection. Terbinafine may be considered as an adjunctive therapy to voriconazole for its in vitro synergistic effect and possibly improved in vivo efficacy; however, combination with amphotericin B should be avoided because of in vitro antagonistic interactions.