Luliconazole, an alternative antifungal agent against Aspergillus terreus

Luliconazole, an alternative antifungal agent against Aspergillus terreus

Journal de Mycologie Médicale (2017) 27, 351—356 Available online at ScienceDirect ORIGINAL ARTICLE/ARTICLE ORIGINAL Lulicon...

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Journal de Mycologie Médicale (2017) 27, 351—356

Available online at



Luliconazole, an alternative antifungal agent against Aspergillus terreus M. Zargaran a,b, S. Taghipour b, N. Kiasat b, E. Aboualigalehdari b, A. Rezaei-Matehkolaei a,b, A. Zarei Mahmoudabadi a,b,*, F. Shamsizadeh b a

Infectious and tropical diseases research center, health research institute, Ahvaz Jundishapur university of medical sciences, Ahvaz, Iran b Department of medical mycology, school of medicine, Ahvaz Jundishapur university of medical sciences, Ahvaz, Iran Received 14 March 2017; received in revised form 9 April 2017; accepted 10 April 2017 Available online 5 May 2017

KEYWORDS Aspergillus terreus; Amphotericin B; Caspofungin; Posaconazole; Fluconazole; Luliconazole; Voriconazole

Summary Aspergillus terreus is the fourth leading cause of invasive and non-invasive aspergillosis and one of the causative agents of morbidity and mortality among immunocompromised and high-risk patients. A. terreus appears to have increased as a cause of opportunistic fungal infections from superficial to serious invasive infections. Although, invasive aspergillosis is often treated empirically with amphotericin B, most A. terreus isolates are resistant both in vivo and in vitro to some antifungal drugs. In this study, we aimed to evaluate antifungals susceptibility profiles of the different strains of A. terreus against amphotericin B, caspofungin, fluconazole, voriconazole, posaconazole and luliconazole. Forty A. terreus strains originating from environmental sources (air and soil) were identified using by macroscopic and microscopic features. Six antifungals including, amphotericin B, caspofungin, fluconazole, voriconazole, posaconazole and luliconazole were applied for susceptibility tests. Our results show that tested isolates had different susceptibility to antifungals. The lowest MICGM related to luliconazole (0.00236 mg/ ml), followed by posaconazole (0.18621 mg/ml), voriconazole (0.22925 mg/ml), caspofungin (0.86 mg/ml), fluconazole (8 mg/ml) and amphotericin B (11.12 mg/ml). This study demonstrated that luliconazole had an excellent in vitro activity against all tested isolates of A. terreus, with MICGM 0.00236 mg/mL than other tested antifungals. As a result, luliconazole could be a possible alternative antifungal for the treatment of aspergillosis due to A. terreus. # 2017 Elsevier Masson SAS. All rights reserved.

* Corresponding author at: Infectious and tropical diseases research center, health research institute, Ahvaz Jundishapur university of medical sciences, Golestan Dist, Esfand St., Ahvaz 61357-15794, Khuzestan, Iran. E-mail address: [email protected] (A. Zarei Mahmoudabadi). 1156-5233/# 2017 Elsevier Masson SAS. All rights reserved.


Introduction Aspergillus terreus is an opportunistic fungus that has been described as one of the causative agent of aspergillosis in the recent years [1]. For example, endocarditis [2], spondylodiscitis [3], pulmonary aspergillosis, endophthalmitis [4,5], onychomycosis [6], otomycosis [7], osteomyelitis, aspergillosis of bone and joint [8], mycotic aneurysm [9], mycocarditis [10] and keratitis [11]. A. terreus is classified as a hyaline mold with widely existence in throughout the world in soil, air and vegetable materials. Furthermore, tropical and sub-tropical areas are presenting favorable conditions for growing A. terreus [6,11]. Invasive aspergillosis is a life-threatening infection with high mortality in immunocompromised patients. The mortality rate of disease is high, about 85% and around 50% if treated with antifungals [12]. A. terreus causes of a range of diseases including, superficial, subcutaneous and systemic infections as well as allergic reactions [2,11,13]. Invasive infections by this species make up about 4% of all invasive aspergillosis and the mortality rates are higher than other species of Aspergillus [1]. Amphotericin B, voriconazole, and posaconazole are classical antifungal drugs, which commonly use in the treatment of aspergillosis [14]. Usually, in clinic, voriconazole is prescribed as the first-line antifungal therapy for invasive aspergillosis, however aspergillosis infections with A. terreus have been failed in 52.9% of cases in comparison to 65.3% for other antifungal drugs [1]. Common causative agents aspergillosis are relatively sensitive to amphotericin B, however, intrinsically, resistance to amphotericin B is seen in A. terreus [15]. Resistance to amphotericin is due to differences in quality and quantity of membrane lipid compositions (especially ergosterol) [16]. In contrast, a recent study showed that resistance to amphotericin is not associated with the cell membrane ergosterol [17]. Posaconazole was discussed as an antifungal choice for prophylaxis in high-risk patients [18]. However, fluconazole is as one of the safest antifungals for the therapy of fungal infections, but because of increased resistance in fungi it has limited its use [19]. Furthermore, it has been demonstrated that Aspergillus species have intrinsic resistance to fluconazole [20—22]. Recently, caspofungin (Cancidas) was used for the treatment of invasive aspergillosis and candidiasis especially in AIDs patients [17,23—25]. Cancidas is comparable in efficacy and safety with other antifungals for invasive mycosis as well as less toxic than amphotericin B [17,25]. Luliconazole (C14H9Cl2N3S2) is an imidazole antifungal drug that approved for the treatment of cutaneous mycosis in Japan from 2005 as 1% cream (Luzu) [26]. Drug is highly effective against dermatophytes and Candida species in vitro [26,27]. On the other hand, luliconazole has excellent tolerability and no systemic side effects were reported when used as topical preparation [28,29]. However, in a few cases skin erythema was reported [30]. Although, luliconazole recently was more used for the treatment of dermatophytosis, researches show that it is also effective against Aspergillus species [31,32]. Luliconazole is newly under evaluation by researchers for Aspergillus species and some studies have shown that luliconazole has significantly effect on A. fumigatus [32].

M. Zargaran et al. In the present study, we aimed to evaluate antifungals susceptibility profile of different environmental strains of A. terreus against six antifungal drugs including, amphotericin B, caspofungin, fluconazole, voriconazole, posaconazole and luliconazole.

Materials and methods Collocation of A. terreus strains and identification A. terreus strains were captured from air and soil samples. All samples were inoculated on Sabouraud dextrose agar (SDA) (Merck, Germany) and incubated at ambient temperature for at least one week. All suspected A. terreus isolates were detected by morphological and microscopy features. Colonies of A. terreus on SDA were velvety, cinnamon to brown with radial folds. The microscopic morphology of A. terreus including, long conidiophores, globose vesicles, compact, columnar and biseriate phialides with globose to ellipsoidal conidia. Moreover, the laterally attached aleurioconidia (3—5 mm in diameter) to vegetative mycelium are the main identical characteristic of A. terreus (Fig. 1) [33,34].

Antifungals stock preparation A stock solution of caspofungin (Sigma—Aldrich, Germany) 1.25 mg/ml, amphotericin B (Sigma—Aldrich, Germany) 32 mg/ml, fluconazole (Serva, USA) 32 mg/ml, luliconazole (APIChem Technology, China) 80 mg/ml, posaconazole (Sigma—Aldrich, Germany) 1.75 mg/ml and voriconazole


Figure 1 Colony and microscopy morphology of Aspergillus terreus (arrow shows aleurioconidia).

Luliconazole against A. terreus [(Figure_2)TD$IG]

Figure 2


Resazurin dye test for determining MIC of luliconazole to Aspergillus terreus strains.

(Sigma—Aldrich, Germany) 1.25 mg/ml was prepared in dimethyl sulfoxide (DMSO, Fluka, Germany). Stock solutions were kept at room temperature for complete dissolving and then stored at 20 8C until use.

Resazurin solution A stock solution of 0.01% of Resazurin (Sigma—Aldrich, Germany) was prepared in sterile distilled water, mixed completely, sterilized by the membrane filter and stored in a brown bottle at 4 8C. Then, 1 ml of stock solution was added to 9 ml of RPMI 1640 (Bio IDEA, Iran) for preparing working RPMI 1640, Resazurin media.

Preparation of standard suspension A. terreus strains were cultured on SDA and incubated at 29 8C for 48—72 h. Then, a spore suspension of each strain was prepared in sterile 0.85% saline containing 1% of Tween 20 (Merck, Germany) and adjusted to 0.5 McFarland standard at wavelength 520 nm (Transmittance 80—82). Finally, standard suspensions were diluted with working RPMI 1640, Resazurin media [35].

Antifungal assay Minimum inhibitory concentrations (MICs) for A. terreus strains were determined by the CLSI M 38-A broth microdilution methodology according to Guinea et al., study [36]. A serial dilution of each antifungal agent was prepared as follows; caspofungin from 8 to 0.0156 mg/ml, amphotericin B from 128 to 0.25 mg/ml, fluconazole from 128 to 0.25 mg/ ml, posaconazole from 32 to 0.0625 mg/ml, voriconazole from 8 to 0.0156 mg/ml, and luliconazole from 2 to 0.0009 mg/ml. Each serial dilution of antifungal drugs was

prepared in working RPMI 1640, Resazurin media. Then, 100 ml of each A. terreus suspension and 100 ml of each antifungal serial dilution were added to each microplate well. Finally, microplates were incubated at 35 8C for 96 h in humid incubator. Microplates were read after 72 and 96 h and the last well remained blue in color (no grow for microorganisms) was defied MIC for each strain of A. terreus (Fig. 2). In the present study MIC50, MIC90, MICGM and epidemiological cutoff values (ECVs) were calculated according previous studies [37,38].

Results The results of the in vitro antifungal susceptibility tests of six antifungal agents against 40 strains of A. terreus are summarized in Table 1. As shown in Table 1, only one isolate of A. terreus was resistant (MIC  4 mg/ml) to voriconazole after 72 h that increased into two isolates after 96 h and the rest of them were sensitive in MICs 0.0625—2 mg/ml. Seventeen of 40 A. terreus strains (42.5%) were inhibited at the concentration of 0.0625 mg/ml of voriconazole, whereas 62.5% of isolates inhibited at the same concentration of posaconazole. Totally, luliconazole, posaconazole, voriconazole, and caspofungin were respectively demonstrated that have good activities against all tested isolates. As shown, the 100% of isolates were inhibited at the concentration of 0.0625 mg/ml or lower than this MIC. Ninety-five percent of isolates had MICs equal or lower than epidemiologic cut-off values (ECV), for voriconazole, followed by posaconazole (77.5%), amphotericin B (47.5%) and caspofungin (17.5%) (Table 2). But not yet, not defined ECV for fluconazole and luliconazole. The lowest MICGM related to luliconazole (0.00236 mg/ml), followed by posaconazole (0.18621 mg/ml), voriconazole (0.22925 mg/ml),

354 Table 1

M. Zargaran et al. MIC distributions of six antifungals for 40 strains of Aspergillus terreus.


Time Antifungal concentrations (mg/ml) (h) 0.0009 0.0019 0.0039 0.0078 0.0156 0.0312 0.0625 0.125 0.25 0.5 1.0 2.0 4.0 8.0 16 32 64 128

Amphotericin 72 B 96 Fluconazole 72 96 Luliconazole 72 96 Caspofungin 72 96 Voriconazole 72 96 Posaconazole 72 96










* * * 15 4 * * * * * *

* * * 8 0 * * * * * *

* * * 7 5 * * * * * *

* * * 9 10 * * * * * *

* * * 1 9 4 0 0 0 * *

* * * 0 10 0 0 0 0 * *

* * * 0 2 2 2 17 17 25 25

* * * 0 0 1 0 0 0 0 0

0 11 2 0 0 0 1 3 1 6 0



8 3

0 3 3 0 2 1 0 6 0 0 0 0 0 0 0 5 14 5 1 4 8 14 4 1 2 3 15 2 2 0 8 0 2

2 3 2 * * 7 20 1 0 2 2






1 5 2 * * 2 4 0 2 1 0

0 0 1 * * * * * * 0 1

1 2 1 * * * * * * 2 2

2 2 4 * * * * * * * *

28 14 22 * * * * * * * *

*: not tested.

Table 2

In vitro susceptibilities of 40 strains of Aspergillus terreus against six antifungals.

Antifungal agents

Amphotericin B Caspofungin Fluconazole Voriconazole Posaconazole Luliconazole

Time (h)

72 96 72 96 72 96 72 96 72 96 72 96

%  ECV a

MIC (mg/ml) MIC range




128—0.25 128—1 8—0.015 8—0.062 128—0.25 128—0.25 4—0.0625 8—0.0625 32—0.0625 32—0.0625 0.0156—0.0009 0.0625—0.0009

8 128 1 4 8 128 0.25 0.5 0.0625 0.0625 0.0019 0.0156

128 128 4 4 128 128 1 2 4 4 0.0009 0.0039

11.12 47.66 0.86 2.62 8 28.3 0.22925 0.41323 0.18621 0.21764 0.00236 0.01057

47.5 20 17.5 7.5 ND a ND 95 57.5 77.5 62.5 ND ND


% of MICs  ECV (ECV = 4 mg/ml for amphotericin B, 0.25 mg/ml for caspofungin, 1 mg/ml for voriconazole, 0.25 mg/ml for posaconazole). No ECVs were available for fluconazole and luliconazole.

caspofungin (0.86 mg/ml), fluconazole (8 mg/ml) and amphotericin B (11.12 mg/ml).

Discussion A. terreus is one of the species of Aspergilli, as a saprophytic fungus that grow in soil and vegetative materials as well as airborne spores. Although, aspergillosis is usually caused by A. niger, A. fumigatus and A. flavus [7,8,39], it is believed that clinical incidence of A. terreus has increased in recent years [6]. Literatures have shown that A. terreus caused different types of aspergillosis, such as onychomycosis [6], keratitis [11], endophthalmitis [5], endocarditis [2], ureteral obstruction [40], bones and joints [8]. The susceptibility of A. terreus to amphotericin B have decreased both in vitro and in vivo [1,6,11,41] as well as in a murine model with disseminated aspergillosis [16]. On the other hand, some researchers have believed that A. terreus

is intrinsically resistant to amphotericin B [1]. Elefanti et al., were reported the clinical breakpoints of amphotericin B for A. terreus as,  0.25 mg/ml susceptible, 0.5 mg/ml intermediate, and  1 mg/ml resistance [42]. According to Elefanti et al., breakpoints, we found that only 2(5%) and 38 (95%) of our isolates were susceptible and resistant to amphotericin B, respectively. Therefore, our results are consistent with other studies that reported A. terreus is often resistance to amphotericin B [21,34,38]. In contrast, in a study by Fernandez et al., clinical and environmental isolates of A. terreus had a MIC range 1—8 mg/ml with a % < ECV 94.74 and 95.24, respectively [13]. Whereas, % < ECV for our isolates was 47.5. Although, the CLSI not determined any clinical breakpoints for A. terreus, ECVs were defined for amphotericin B ( 4 mg/ml) [38]. Several clinical and experimental studies were reported effective therapeutic effects of voriconazole, posaconazole and caspofungin against invasive aspergillosis caused by

Luliconazole against A. terreus A. terreus [2,34,39—41,43,44]. Furthermore, voriconazole and posaconazole were recommended for the treatment and prophylaxis of invasive aspergillosis, respectively [36,39]. Guinea et al. were classified Aspergillus resistance for voriconazole as an MIC  4 mg/ml [36]. They claimed that 400 clinical strains of Aspergillus species collected from 1999 to 2007 were fully susceptible to voriconazole. We detected only one voriconazole resistance A. terreus, and our study compatible with Guinea et al. [36], Fernandez et al. [13], studies that 100% of examined isolates were sensitive. Several reports show that caspofungin was evaluated as first-line therapy for invasive aspergillosis and candidiasis in hematological diseases, neutropenic and non-neutropenic patients due to low adverse effects and high efficacy [17,25,39]. Aspergillosis due to A. terreus was successfully treated with caspofungin [45]. Furthermore, two clinical strains of A. terreus were tested against caspofungin using microdilution method by Gupta et al., [14]. In this study, both strains had MIC = 0.06 mg/ml whereas, in our study only 15% of tested strains showed this quail or lower MICs. Although, fluconazole is a safe antifungal for some types of fungal infections (Candidiasis), however high MICs were reported for Aspergillus species in vitro [46]. As a result, it is found that fluconazole does not have clinical efficacy against Aspergillus species. Our findings were also confirmed previous finding with high MIC range for the most tested isolates. In clinic, posaconazole is described as prophylaxis and an alternative to salvage therapy for invasive aspergillosis [47—49]. Furthermore, all A. terreus strains in a study were sensitive to posaconazole with MIC of  1 mg/ml [41]. Although, the CLSI not determined any clinical breakpoints for A. terreus, ECVs were defined for caspofungin ( 0.25 mg/ml), voriconazole (1 mg/ml) and posaconazole (0.25 mg/ml) [50,51]. In addition, based on MIC50 and MIC90, luliconazole (0.0019 and 0.0009 mg/ml, respectively) compared to other antifungals is highly less than, so demonstrated that luliconazole has potent activity against all A. terreus isolates. In conclusion, this study showed that in vitro antifungal activity of luliconazole is stronger than other drugs with lowest MICGM (0.00236 mg/ml) against A. terreus, followed by posaconazole (0.18621 mg/ml) and voriconazole (0.22925 mg/ml). Due to availability of luliconazole as topical formulation, it could be potency for cutaneous aspergillosis. In addition, drug has excellent pharmacokinetic characteristics including, well tolerability, both fungicidal and fungistatic activities, and low toxicity. Regarding to these futures, we proposed that luliconazole could be a possible alternative antifungal for the treatment of invasive aspergillosis in the future. However, systemic formulation of luliconazole must be prepared and tested in animal model for safety.

Disclosure of interest The authors declare that they have no competing interest.

Acknowledgments We would like to thank the department of medical mycology, Ahvaz Jundishapur University of Medical Sciences for their

355 support. Furthermore, some used antifungal drugs and culture media obtained from for other projects (No. OG93122, OG-94139).

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