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A collection of azole-susceptible (n = 141) and azole-resistant (n = 27) Aspergillus fumigatus isolates was tested against seven antifungal drugs, including the new imidazoles lanoconazole and luliconazole. The luliconazole and lanoconazole MIC90 values for the azole-susceptible strains were 0.001 μg/ml and 0.008 μg/ml, and those for the azole-resistant strains were 0.016 μg/ml and 0.032 μg/ml.
Invasive aspergillosis caused by Aspergillus fumigatus is a difficult-to-diagnose, life-threatening opportunistic fungal infection associated with significant morbidity and mortality (1). Survival rates improved significantly after the introduction of triazole antifungal agents such as voriconazole (2,–4). However, triazole-resistant Aspergillus fumigatus strains are emerging worldwide due to long-term triazole therapy or, more commonly, are selected in the environment through exposure to azole fungicides (4,–7). Recently, a new antifungal agent, isavuconazole, was shown to be noninferior to voriconazole for treatment of infections caused by Aspergillus species (8). However, cross-resistance exists for azole-resistant Aspergillus. Luliconazole, a topically related compound of lanoconazole, has been approved by the FDA for topical treatment of tinea cruris, tinea corporis, and tinea pedis. Luliconazole had neither clastogenic or mutagenic effects in genotoxicity tests, and no effect on fertility or reproductive function was noted. Luliconazole affects ergosterol biosynthesis by inhibiting the azole target protein lanosterol 14α-demethylase (cyp51A), which is the key enzyme that catalyzes the oxidative removal of the 14α-methyl group of lanosterol to give 14-15-desaturated intermediates in ergosterol biosynthesis (9). Recently, in vitro antifungal susceptibility testing of lanoconazole and luliconazole demonstrated potent efficacy against Trichophyton rubrum and Epidermophyton floccosum (9,–14). In addition, animal studies and small human series suggested that luliconazole and lanoconazole are effective in treating dermatophytosis and onychomycosis (15, 16). Only limited data on the in vitro activity of lanoconazole and luliconazole against Aspergillus species are available. Therefore, the aim of the present study was to investigate the in vitro activity of these two new imidazoles and five comparators against a large collection of azole-susceptible and -resistant A. fumigatus strains with various point mutations from clinical and environmental sources. A total of 168 well-characterized A. fumigatus strains from the culture collection of the Invasive Fungi Research Center (IFRC) were included. Azole-susceptible (n = 141) and -resistant (n = 27) strains originated from nail, sputum, bronchoalveolar lavage, sinus discharge, and skin biopsy samples. Environmental samples came from soil and air samples. Most of the azole-resistant A. fumigatus strains (n = 10) harbored a leucine-to-histidine substitution at codon 98, along with a 34-bp tandem repeat in the cyp51A promoter region, but TR46/Y121F/T289 (n = 2) and other point mutations (n = 8) such as G54, M220, G138C, and G432C were also included. Resistant isolates without mutations in cyp51A were also included (n = 7). MICs were determined based on CLSI M38-A2 (17). Concentration ranges of 0.001 to 1 μg/ml for luliconazole (Nihon Nohyaku Co, Osaka, Japan) and lanoconazole (Nihon Nohyaku Co.), 0.016 to 16 μg/ml for itraconazole (Janssen, Beerse, Belgium), voriconazole (Pfizer, Sandwich, United Kingdom), and amphotericin B (Bristol-Myers-Squib, Woerden, The Netherlands), and 0.008 to 8 μg/ml for posaconazole (Merck Sharp & Dohme BV, Haarlem, The Netherlands) and caspofungin (Merck Sharp & Dohme BV) were used. Stock solutions were prepared in dimethyl sulfoxide. Conidial suspensions were prepared by scraping the surface of fungal colonies with a sterile cotton swab moistened with physiological saline solution containing 0.05% Tween 40 and were adjusted to optical densities ranging from 0.09 to 0.11 (0.5 × 106 to 3.1 × 106 CFU/ml) measured at 530 nm. Inoculum suspensions, including mostly nongerminated conidia, were diluted 1:50 in RPMI 1640 medium, and the final inoculum in assay wells was between 0.4 × 104 and 5 ×104 CFU/ml. Microdilution trays were incubated at 35°C for 48 h. MICs were determined visually as the lowest concentration which provided complete inhibition of growth, while minimum effective concentrations (MECs [caspofungin only]) were determined microscopically as the lowest concentration of drug promoting the growth of small, round, compact hyphae relative to the appearance of the filamentous forms seen in the control wells. Candida krusei (ATCC 6258) and Paecilomyces variotii (ATCC 3630) were included as quality controls (17). All tests were performed in duplicate, and differences of the mean values were determined by using Student's t test with the statistical SPSS package (version 7.0). P values of <0.05 were considered statistically significant. Table 1 summarizes the in vitro susceptibility of 168 susceptible and resistant A. fumigatus isolates. The novel imidazoles luliconazole and lanoconazole demonstrated potent activity against all A. fumigatus isolates, in comparison to voriconazole, itraconazole, and posaconazole. MICs of lanoconazole and luliconazole against all A. fumigatus isolates ranged from <0.001 to 0.5 μg/ml and from <0.001 to 0.016 μg/ml, respectively, compared to 0.064 to >16 μg/ml for itraconazole, 0.064 to >16 μg/ml for voriconazole, and 0.008 to 8 μg/ml for posaconazole. The lanoconazole and luliconazole geometric mean (GM) MICs against all isolates were 0.0024 μg/ml and 0.0012 μg/ml, respectively, while those of the other agents were as follows: itraconazole, 0.4243 μg/ml; voriconazole, 0.2555 μg/ml; posaconazole, 0.0968 μg/ml; caspofungin, 0.0535 μg/ml. Basically, the GM MIC value of luliconazole against all A. fumigatus isolates was 2 log2 dilutions lower than that of lanoconazole. However, no statistically significant (P > 0.05) differences in the lanoconazole and luliconazole susceptibility patterns were detected between strains. MICs of luliconazole and lanoconazole for the resistant isolates with various point mutations in the cyp51A gene were approximately similar to those of the susceptible isolates, but strains with TR46/Y121F/T289 mutations showed less susceptibility, with a 4-log2-dilution step compared to the other resistant strains harboring TR34/L98H, G54, M220, G138C, and G432C (Table 2).
In previous studies, we demonstrated that the prevalence of azole-resistant A. fumigatus with predominant TR34/L98H mutations in the cyp51A gene in Iran has increased remarkably from 3.3% to 6.6% (6, 7). Treatment regimens of Aspergillus infections with triazole agents are associated with a poor outcome when azole resistance is involved (18). In this study, molecularly identified strains of A. fumigatus strains from both clinical and environmental sources were subjected to antifungal susceptibility testing with newer imidazoles.
Recently, several studies have shown high in vitro and in vivo efficacy of luliconazole against a limited number of dermatophytes and other agents causative of onychomycosis (11,–14, 19, 20). In addition, in the present study, lanoconazole and luliconazole showed potent activity against the wild-type strain as well as against azole-resistant mutants of A. fumigatus, but high MIC values for lanoconazole against two isolates with TR46/Y121F/T289 were observed. While the most common TR34/L98H mutation confers resistance to all azoles, TR46/Y121F/T289A confers resistance to voriconazole and isavuconazole but shows a variable influence on the MICs of itraconazole and posaconazole. The point mutations G54 and M220 in cyp51A induce resistance mainly to itraconazole and posaconazole (21). Only limited data are available on the effect and related toxicity of systemic use of luliconazole and lanoconazole. Oral luliconazole therapy in a murine model of invasive aspergillosis was superior to that of itraconazole, and intravenous luliconazole appeared to be highly effective in comparison with intravenous amphotericin B (22). In this study, 90% of the animals stayed alive when 2.5 mg/kg of body weight/day of luliconazole was used whereas only 30% of animals survived when amphotericin B (5 mg/kg/day) was used (22).
The current study demonstrated that the in vitro antifungal activities of luliconazole and lanoconazole against susceptible and resistant A. fumigatus isolates are apparently superior to those of polyenes, other azoles, and echinocandins. Clinical effectiveness in the treatment of Aspergillus infection and pharmacodynamic assessment and development of epidemiologic cutoff values (ECVs)/breakpoints remain to be established. In conclusion, we suggest that these two new imidazoles are promising candidates for treatment of invasive aspergillosis caused by either azole-susceptible or -resistant isolates.
This study was supported financially by a grant from the School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran (no. 1814), which we gratefully acknowledge.
We are grateful to Iman Haghani for excellent technical assistance and help with antifungal susceptibility testing.
J.F.M. received grants from Astellas, Basilea, and Merck. He has been a consultant to Astellas and Merck and has received speaker's fees from Gilead Sciences, Merck, Pfizer, and United Medical. The rest of us have no conflicts of interest to declare.