Journal de Mycologie Médicale (2018) 28, 374—378

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ORIGINAL ARTICLE/ARTICLE ORIGINAL

Luliconazole, a new antifungal against

Candida species isolated from different
sources
S. Taghipour a, N. Kiasat a, S. Shafiei a, M. Halvaeezadeh a,
A. Rezaei-Matehkolaei a,b, A. Zarei Mahmoudabadi a,b,*

a
Department of Medical Mycology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences,
Ahvaz, Iran
b
Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University
of Medical Sciences, Golestan BLV, Ahvaz, Iran

Received 13 April 2017; received in revised form 9 November 2017; accepted 13 November 2017
Available online 29 November 2017

KEYWORDS Summary
Luliconazole; Objective. — Luliconazole is an inhibitor for sterol 14-a-demethylase in fungal cells with a broad-
Antifungal; spectrum antifungal activity against dermatophytes, Candida albicans, Malassezia species, dema-
Candida albicans; tiaceous and hyaline hyphomycetes. Furthermore, luliconazole has been clinically used for the
Mucocutaneous treatment of pityriasis versicolor, dermatophytosis, onychomycosis, cutaneous and mucocutane-
candidiasis ous candidiasis. In the present study, we aimed to evaluate in vitro antifungal activity of
luliconazole against several strains of Candida species recovered from different clinical materials.
Materials and methods. — In the present study, 104 strains of Candida species including, 34 isolates
from vaginitis, 23 isolates from AIDS patients with vaginal candidiasis, 24 isolates from neutropenic
patients and 24 isolates from tracheal tubes, were examined for susceptibility tests. A serial
dilution of luliconazole (4—0.008 mg/mL) was tested against different strains of Candida species
recovered from different sources.
Results. — The minimum inhibitory concentration (MIC) range and MIC90 of vaginal isolates (HIV )
were 1—0.063 and 1 mg/mL. Furthermore, the most of strains (50%) had a MIC of 0.5 mg/mL. The MIC
ranges were similar (2—0.016 mg/mL) for both vaginal (HIV+) and neutropenic patients isolates,
whereas, MIC90 for them were 0.5 and 1 mg/mL, respectively. All tracheal tubes strains were
inhibited at the range of 2—0.008 mg/mL with MIC90 = 1 mg/mL. Totally, the lowest MIC50
(MIC = 0.015 mg/mL), MIC90 (MIC = 1 mg/mL) and MICGM (MIC = 0.05 mg/mL) are correlated to
C. glabrata, a non-albicans species.

* Corresponding author. Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of
Medical Sciences, Ahvaz, Iran.
E-mail address: zarei40@hotmail.com (A.Z. Mahmoudabadi).

https://doi.org/10.1016/j.mycmed.2017.11.004
1156-5233/# 2017 Elsevier Masson SAS. All rights reserved.
Luliconazole against Candida species
Conclusion. — It is concluded that, luliconazole could be an alternative anti-Candida agent,
however, in vivo studies must be confirmed usefulness of drug for clinical usage.
# 2017 Elsevier Masson SAS. All rights reserved.
Introduction
Luliconazole, (-)-(E)-((4R)-4-(2,4-dichlorophe-nyl)-1,3-
dithiolan-2-ylidene) (1H-imidazol-1-yl) aceto-nitrile), is a
novel imidazole with a unique chemical structure (Fig. 1)
[1]. It is an inhibitor for sterol 14-a-demethylase (cyto-
chrome P45014DM, CYP51) in fungal cells during ergosterol
biosynthesis [2]. The process led to reduction of ergosterol
and accumulation of 14-a-methyl sterols in fungal cells and
finally cause cell death [1]. It has a broad-spectrum anti-
fungal activity against dermatophytes [3,4], Malassezia spe-
cies [5], dematiaceous and hyaline hyphomycetes [6] and
Candida albicans [7]. Furthermore, many clinical researches
are indicated that luliconazole has excellent efficacy for the
treatment of dermatophytic onychomycosis [1], tinea pedis
[8], cutaneous candidiasis [9], mucocutaneous candidiasis
[10] and pityriasis versicolor [9]. Luliconazole was launched
in 2005 in Japan as 1% cream and solution for fungal infection
therapy [3]. Furthermore, it has been approved in India in
2010 as topical antifungal for the treatment of cutaneous
mycosis [11].
Both fungistatic and fungicidal activities for luliconazole
against dermatophytes were documented in literatures [1].
Luliconazole is highly active against C. albicans and derma-
tophytes in vitro at range 0.031—0.13 mg/mL [3] and 0.001—
0.625 mg/mL [12], respectively. Candida species are a
mycoflora of human body and causative agents of a wide
range of opportunistic infections (candidiasis). During seve-
ral last decades, an increasing in resistant to antifungals was
found among non-albicans species as well as albicans species
[13,14]. Recently, Abastabar et al. tested luliconazole
against azole susceptible and resistant strains of Aspergillus
fumigatus and found that all strains are sensitive to lulico-
nazole at the range of  0.001—0.016 mg/mL [15]. Further-
more, we have previously shown that A. terreus isolates
highly sensitive to luliconazole in vitro [16].
Although, there are considerably literatures in related to
use luliconazole for dermatophytosis therapy, few data indi-
cated the efficacy of drug against strains of Candida species.
As a result, in the present study, we aimed to evaluate in
Figure 1 Chemical structure of luliconazole (C14H9Cl2N3S2).
375
vitro antifungal activity of luliconazole against several
strains of Candida species recovered from different clinical
materials.
Materials and methods
Organisms
In the present study, 104 strains of Candida species were
examined for in vitro antifungal susceptibility test against
luliconazole. All strains were previously isolated from
different clinical sources including; 34 Candida isolates
from patients with vulvovaginal candidiasis, 23 Candida
isolates from AIDS patients with vaginal candidiasis, 24
isolates from oral secretions of patients with neutropenia
and 24 Candida isolates from tracheal tubes of hospitali-
zed patients in ICUs (Table 1). Strains were primarily
identified using several phenotypic methods including,
germ tube test, growth at 45 8C, chlamydoconidia forma-
tion in Cornmeal agar — Tween 80 and colored colonies
morphology on CHROMagar Candida medium. In addition,
molecular methods such as PCR-RFLP and sequencing
methods were also performed. All C. albicans strains were
identified using above mentioned classical methods and
non—albicans Candida isolates detected using PCR and
PCR-RFLP methods.
In this study, C. parapsilosis (ATCC 22019), C. albicans
(NBRC 0917) were used as controls for antifungal suscepti-
bility.
Preparation of luliconazole
A stock solution of luliconazole (APIChem Technology, China)
was prepared at the concentration of 320 mg/mL using
dimethyl sulfoxide, DMSO (Merck, Germany). Stock was
maintained in a dark bottle and kept at 20 8C.
Standard suspension
A standard inoculum suspension of each Candida strains was
prepared from an overnight yeast culture in accordance with
Clinical and Laboratory Standard institute (CLSI). Briefly, a
suspension of a small colony was prepared in distilled water
and adjusted to 0.5 McFarland standard by a spectrophoto-
meter [17].
Antifungal susceptibility testing
Microdilution method (96 wells microplate) using colorime-
tric techniques was used for antifungal susceptibility testing
against microorganisms [18—21]. A concentration of lulico-
nazole antifungal was prepared in the range of 4 to 0.008 mg/
mL using by RPMI 1640 (Bio-Idea, Iran) with % 0.01 resazurin
376 S. Taghipour et al.

Table 1 The frequency of Candida species used for antifungal susceptibility.

Species Isolates sources Total

Vulvovaginal Candidiasis Neutropenic patients ICU
HIV+ HIV patients
C. albicans 15 (14.4%) 30 (28.8%) 7 (6.7%) 3 (2.9%) 55 (52.9%)
C. glabrata 4 (3.8%) 3 (2.9%) 4 (3.8%) 5 (4.8%) 16 (15.4%)
C. tropicalis 0 (0.0%) 0 (0.0%) 5 (4.8%) 9 (8.6%) 14 (13.5%)
C. krusei 1 (0.9%) 0 (0.0%) 4 (3.8%) 7 (6.7%) 12 (11.5%)
C. dubliniensis 3 (2.9%) 0 (0.0%) 1 (0.9%) 0 (0.0%) 4 (3.8%)
C. kefyr 0 (0.0%) 1 (0.9%) 1 (0.9%) 0 (0.0%) 2 (1.9%)
C. lambica 0 (0.0%) 0 (0.0%) 1 (0.9%) 0 (0.0%) 1 (0.9%)
Total 23 (22.1%) 34 (32.7%) 23 (22.1%) 24 (23.1%) 104 (100%)

(Sigma — Aldrich, Germany). Then, 100 mL of each serial
dilution of luliconazole was added to the wells and seeded with
100 mL of diluted standard suspension (1:50). One well contain-
ing inoculum suspension was considered as a positive control
and negative control contained the RPMI 1640 with resazurin.
All microplates were incubated at 35 8C for 24—48 hours. The
lowest concentration of the luliconazole that maintained the
blue color of the colorimetric indicator was determined as the
minimum inhibitory concentration (MIC) for each strain.
Results
Vaginal isolates (HIV )
Thirty-four strains of Candida including, C. albicans (30),
C. glabrata (3) and C. kefyr (1) were evaluated for lulico-
nazole susceptibility in vitro. Minimum inhibitory concentra-
tion range of luliconazole for tested strains was 4—0.063 mg/
mL. As shown in Table 2, after 24 h, the MIC of the most of
strains (50%) was 0.5 mg/mL, followed by 38.2% of isolates
1 mg/mL. The rest of them (11.8%) had MIC = 0.063 mg/mL.
Vaginal isolates (HIV+)
patients, are presented in Table 2. The most of tested
isolates (91.3%) were inhibited at the concentration of lower
than 0.5 mg/mL of luliconazole.
ICU isolates
Twenty-four strains of C. albicans (3), C. glabrata (5),
C. tropicalis (9) and C. krusei (7) were evaluated against a
serial dilution of luliconazole (2—0.008 mg/mL) (Table 2). All
isolates were recovered from tracheal tubes from hospita-
lized patients in ICUs. Minimum inhibitory concentration for
37.5% of strains was 0.008 mg/mL. The most of non-albicans,
exception C. glabrata, were inhibited at the concentration
of 1—0.25 mg/mL of luliconazole.
Neutropenic isolates
Twenty-three strains of different species of Candida from
neutropenic patients were examined for susceptibility test-
ing to luliconazole in vitro. The diversity of MIC ranges for
neutropenic isolates of Candida strains were limited to 2—
0.016 mg/mL and the most of isolates (69.6%) inhibited at the
range of 1—0.5 mg/mL (Table 2).

The MICs of luliconazole against 15 strains of C. albicans, 4 Table 3 MIC range, MIC50, MIC90, MICGM of luliconazole for
strains of C. glabrata, 3 strains of C. dubliniensis and 1 strain 104 tested Candida strains.
of C. krusei, recovered from vaginal lesions of HIV positive
All isolates Time MIC ranges MIC50 MIC90 MICGM
(h) (mg/mL)
Table 2 MIC range, MIC50, MIC90, MICGM of luliconazole for
C. albicans 24 2—0.016 0.5 1 0.46
104 strains of Candida from different sources.
(n = 55) 48 4—0.016 0.5 2 0.58
Isolates sources Time Luliconazole concentrations C. glabrata 24 2—0.008 0.015 1 0.05
(h) (mg/mL) (n = 16) 48 2—0.008 0.015 1 0.05
C. tropicalis 24 2—0.008 0.5 1 0.35
MIC range MIC50 MIC90 MICGM
(n = 14) 48 2—0.008 0.5 2 0.49
Neutropenic (n = 23) 24 2—0.016 0.5 1 0.371 C. krusei 24 1—0.008 0.015 1 0.07
48 2—0.016 0.5 2 0.516 (n = 12) 48 2—0.008 0.25 1 0.11
ICU (n = 24) 24 2—0.008 0.25 1 0.106 C. kefyr 24 0.5—0.063 — — —
48 2—0.008 0.25 1 0.133 (n = 2) 48 1.5—0.063 — — —
Vaginal (HIV+) (n = 23) 24 2—0.016 0.25 0.5 0.160 C. dubliniensis 24 2—0.25 — — —
48 2—0.016 0.25 0.5 0.175 (n = 4) 48 2—0.25 — — —
Vaginal (HIV ) (n = 34) 24 1—0.063 0.5 1 0.670 C. lambica 24 1 — — —
48 4—0.063 1 2 1.250 (n = 1) 48 1 — — —
Total (n = 104) 24 2—0.008 0.5 1 0.256 Total (104) 24 2—0.008 0.5 1 0.26
Neutropenic (n = 23) 48 4—0.008 0.5 2 0.325 48 4—0.008 0.5 2 0.33
Luliconazole against Candida species
Table 3 was indicated the MIC ranges, MIC50, MIC90 and
MICGM for 104 examined strains. As shown, the lowest MIC50
(MIC = 0.015 mg/mL), MIC90 (MIC = 1 mg/mL) and MICGM
(MIC = 0.05 mg/mL) are correlated to C. glabrata, a non-
albicans species.
In this study, MIC for C. albicans (NRBC 0917) and
C. parapsilosis (ATCC 22019) were reported 0.031 and
2 mg/mL, respectively.
Discussion
Luliconazole (imidazole antifungal) has been shown to have
potent activity against several pathogenic fungi (especially
dermatophytes) in vitro [22,23]. Several scientists, espe-
cially Japanese researchers, tested luliconazole for the
treatment of dermatophytosis in clinic and in vitro studies.
Clinical trials were indicated that it has a high efficacy for the
treatment of dermatophytosis [24—26], pityriasis versicolor
[27] and onychomycosis [9]. Shimamura et al. compared the
efficacy of efinaconazole and luliconazole against onycho-
mycosis and found that the last has more prefer to treatment
onychomycosis caused by Trichophyton species [28].
Although, luliconazole was approved by Japanese health
authorities as 1% cream, solution and ointment for superficial
and cutaneous mycoses at 2005, drug is currently under
development in the USA [1]. On the other hand, there are
no more data in drug efficacy on clinical or environmental
isolates of Candida.
In the present study, the efficacy of luliconazole was
studied against different strains of Candida species with
several sources. Minimum inhibitory concentration range
for tested strains was very low, ranked from 2 to
0.008 mg/mL. Niwano et al., [29] and Koga et al., [3] were
the first to report a very low MICs (MIC range: 0.031—0.13 and
0.031—0.25 mg/mL) against C. albicans, respectively. On the
other hand, Niwano et al., were compared an oral regimen of
luliconazole and fluconazole and found that luliconazole was
less effective than fluconazole in a murine model of systemic
candidiasis. [29]. Luliconazole, (1% topical cream) was
completely resolved diaper rash in an infant within one
month without any recurrence after 6 months follow up
[30]. Totally, our study indicated that 91.3% vaginal isolates
from HIV+ have MIC  0.5 mg/mL, followed by tracheal tube
(75%), neutropenic isolates (73.9%) and HIV (61.8%). It is
interesting that the percent of Candida species that had
lower MIC ( 0.5 mg/mL), was included in HIV+ patients. In
contrast, only 61.8% of strains recovered from HIV had the
same MIC. It is suggesting that the isolate sources could be
affected on MIC. It seems that yeasts with low pathogenicity
and high sensitivity with antifungals are more causative
agents in immunocompromised hosts (HIV+, ICU and neutro-
penic patients) than immunocompetent patients.
Due to lack an acceptable breakpoint of luliconazole for
Candida species, the range of sensitivity, dose dependent or
resistance of antifungal is unclear. Totally, the lower MICGM
was 0.05 for C. glabrata followed by, 0.07 mg/mL for
C. krusei, 0.35 mg/mL for C. tropicalis and 0.46 mg/mL for
C. albicans (Table 3). Regarding to these results, the non-
albicans strains were more sensitive to luliconazole than
C. albicans strains. Accordingly, it is concluded that, lulico-
nazole could be an alternative anti-Candida agent. Although,
377
the systemic formulation of luliconazole is unavailable,
however, in vivo studies must be confirmed usefulness of
drug for clinical usage.
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
support. Furthermore, Candida species and culture media
obtained from our previously projects (No: OG-93122, OG-
94139 and OG-94170).
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