Jundishapur J Microbiol. 2020 March; 13(3):e99922.
doi: 10.5812/jjm.99922.
Research Article
Published online 2020 April 5.
Ocular Fungi: Molecular Identification and Antifungal Susceptibility
Pattern to Azoles
Mohammad Soleimani 1, 2 , Zahra Salehi 3 , Azam Fattahi 4, * , Ensieh Lotfali
Ghasemi 7 , Zohreh Abedinifar 2 , Ebrahim Kouhsari 8, 9 , Fardin Ahmadkhani
Mirkalantari 11
5
, Zeynab Yassin 6 , Reza
and Shiva
10
1
Ocular Trauma and Emergency Department, Tehran University of Medical Sciences, Tehran, Iran
Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran
Department of Mycology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
4
Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran
5
Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
6
Antimicrobial Resistance Research Center, Iran University of Medical Sciences, Tehran, Iran
7
Student Research Committee, School of Medicine, Shahid Beheshti University of Medical, Tehran, Iran
8
Clinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Iran
9
Laboratory Sciences Research Center, Golestan University of Medical Sciences, Gorgan, Iran
10
Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
11
Microbiology Department, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran
2
3
*
Corresponding author: Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran. Email:
fattahiazam.mycologist@gmail.com
Received 2019 December 03; Revised 2020 March 18; Accepted 2020 March 20.
Abstract
Background: Treatment of ocular infection by fungi has become a problematic issue, particularly in deep lesion cases, because of
the limited available antifungals and emerging resistance species.
Objectives: The present study was designed for molecular identification and studying the antifungal susceptibility pattern of ocular fungi.
Methods: Fifty-three ocular fungal isolates, including Fusarium spp., Aspergillus spp., yeast spp., and dematiaceous fungi were collected. Initial identification of each sample was performed using routine mycological techniques. ITS1-5.8SrDNA-ITS2 and translation elongation factor (TEF)-1α regions were used for the identification and differentiation of ocular non-Fusarium and Fusarium
fungal species, respectively. The antifungal susceptibility of itraconazole, voriconazole, and posaconazole was determined according to the CLSI guidelines (CLSI M38 and M60, 3rd ed.) for filamentous and yeast species, respectively.
Results: Voriconazole and posaconazole showed excellent activity in all tested isolates; however, some of Fusarium, Aspergillus, and
Curvularia strains showed minimum inhibitory concentration (MIC) ≥ 2 µg/mL. The itraconazole showed different results in all
species, and high MICs (≥ 16 µg/mL) were found.
Conclusions: Finally, in the present study, we tried to identify species involved in fungal ocular infection using the molecular methods, which highlighted the importance of precise identification of species to choose an appropriate antifungal regime. On the other
hand, our findings showed that antifungal susceptibility test is effective to reliably predict the in vivo response to therapy in infections; however, in fungal ocular infection cases, the penetration of antifungals may contribute to predict the outcome.
Keywords: Fungal Ocular Infection, Identification, Fungus Drug Sensitivity Tests
1. Background
Fungal ocular infection (also termed as mycotic keratitis or keratomycosis) is a major ophthalmic issue in tropical and subtropical areas, however, it sometimes presents
in other high-income countries with temperate climates (1,
2). It has known as the main cause of blindness in Asia (3)
and 56% of cases of keratitis were caused by fungi (4). The
most predominant fungal pathogens causing mycotic ker-
atitis are Fusarium spp., Aspergillus spp., and dematiaceous
spp. (2). Fusarium spp. are considered as the major causes
of corneal infection in Iran, Turkey, Taiwan, Thailand, Hong
Kong, Brazil, South Florida, Australia, Tunisia, and Ghana
(5). Treatment of keratitis has become a problematic issue, particularly in deep lesion cases, because of the limited available antifungals and emerging resistance species.
On the other hand, a failure to treat keratitis can result in
vision loss.
Copyright © 2020, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License
(http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly
cited.
Soleimani M et al.
Management of fungal ocular infection is usually
based on the natamycin and topical anti-fungal drugs. Topical amphotericin B 0.3% to 0.5% and voriconazole 1% are
prescribed as alternatives to natamycin (6). Oral and intrastromal injection of voriconazole has also been effective
in cases of a recalcitrant and deep fungal ocular infection
(6, 7). In general, Fusarium spp. have shown the intrinsic
resistance to the most routine medications (8). Natamycin
5% is still considered as the first choice in the treatment
of fungal ocular infection (9), followed by the voriconazole (10). Antifungal resistance pattern caused by fungal
ocular infection is limited; however, in clinics, antifungalresistant fungal species have been reported. It has believed
that the results of clinical response to therapy do not always correspond to in vitro results, which can partly be due
to poor penetration of available remedies resulting in misunderstanding of the precise resistance rate of clinical isolates and also the limitation of standardization of antifungal susceptibility tests in the past (1, 11). Different in vitro
antifungal activities of Fusarium spp. to routine available
antifungals have reported (12-15).
Several studies have reported the in vitro antifungal
susceptibility patterns among fungal isolates causing fungal ocular infection. The treatment takes too long and is
too expensive, and usually, the outcomes are not satisfactory. The significant strategy to reduce the severity and
consequences of the disease is early diagnosis and treatment. Early diagnosis and precise identification of ocular fungal species, as well as the treatment of infected patients, are required to prevent the spread of the disease
and its severe complications. Recently, molecular amplification assays, like polymerase chain reaction (PCR) and sequencing have been reported as highly sensitive and specific methods to identify fungal ocular infection at the
species level.
2. Objectives
The present study was designed for molecular identification and studying the antifungal susceptibility pattern
of ocular fungi.
3. Methods
3.1. Sample Collection and Initial Identification of the Isolates
Fifty-three ocular fungal isolates, including Fusarium
spp. (24), Aspergillus spp. (20), yeast spp. (6), and dematiaceous fungi (5) were collected from the microbiology laboratory of Farabi Eye Hospital, Tehran, Iran in 2018. Initial
identification of each sample was performed by direct microscopy using Gram staining and culture on Sabouraud
2
dextrose agar (SDA; Merck, Germany) at 35°C for at least
seven days. The morphological characteristic of Aspergillus
spp. was studied after seven days of incubation on Czapek
yeast autolysate agar (CYA; Merck, Germany) at 25°C and
35°C. All isolates were stored in tryptic soy broth (TSB; Liofilchem, Italy) at -70°C for further evaluation.
3.2. Molecular Identification
3.2.1. DNA Extraction
The genomic DNA of all strains was extracted from
fresh colony cultured for seven days (for filamentous
fungi), and 24 hours (for Candida spp.) using the isopropanol and proteinase K method (16). Briefly, fungal
spores were scraped and inoculated in 1.5 mL microtubes
with few glass beads; they were frozen quickly in liquid nitrogen, and their cell walls disrupted by pestle. Then, 400
µL of the extraction buffer containing 200 M Tris- HCl (pH
8), 25 mM EDTA, SDS 0.5% w/v, and NaCl 250 mM was added
to the reaction mixture, followed by vortexing for 1 minute,
adding 8 µL proteinase K, and incubating at 55°C for 60
minutes. Then microtubes containing the above mixture
were centrifuged at 1000 ×g for 30 minutes. The liquid
phase was transferred to a fresh tube and the genomic
DNA was extracted using an equal volume of ice- cold isopropanol and the samples were incubated at -20°C for 60
minutes. Next, it was centrifuged at no more than 12500
×g for 10 minutes at 4°C. The supernatant was removed
and the DNA pellet was washed in 300 µL ethanol 70% then
air-dried and resuspended in 50 µL TE buffer (Merck, Germany). DNA was electrophoresed on 1% agarose gel electrophoresis containing DNA safe stain (Simbio, USA) and
visualized under ultraviolet (UV) light. The optical density
of all DNA samples was measured by NanoDrop (ND- 1000,
Thermo Scientific Fisher, US) and stored at -20°C.
3.2.2. PCR Amplification and Sequencing of ITS, TEF- 1α Region
The PCR amplification of ITS1-5.8SrDNA-ITS2 and translation elongation factor (TEF-1α) regions was conducted
for the identification and differentiation of cornea by nonFusarium and Fusarium spp., respectively. A reaction mixture containing 2 µL of the extracted DNA, 12 µL of Taq
DNA Polymerase Master Mix White (Ampliqon), 0.5 µL (10
pm/µL) of the ITS1-forward (5’- TCC GTA GGT GAA CCT GCG
G -3’) and ITS4-reverse (5’- TCC TCC GCT TAT TGA TAT GC3’) (16) and TEF- 1α forward (GTAAGGAGGASAAGACTCACC)
TEF-1α reverse (GTAAGGAGGASAAGACTCACC) primers (designed in this study), and ddH2 O was added to obtain a total volume of 25 µL. The PCR program was designed as follows: initial denaturation for 2 minutes at 95˚C, 35 cycles of
denaturation for 45 seconds at 94˚C, annealing for 45 - 60
seconds (for each primer set) at 58˚C, an extension for 60
Jundishapur J Microbiol. 2020; 13(3):e99922.
Soleimani M et al.
seconds at 72˚C, and a final extension at 72˚C for 7 minutes.
The amplified products were assessed by electrophoresis
on 1.5% agarose gel in TBE buffer (Merck, Germany) and
stained with safe stain (Simbio, USA). The products were
sequenced and analyzed using the non-redundant nucleic
databases BLAST (http://www.ncbi.nim.nih.gov/BLAST/).
3.2.3. PCR Amplification of HWP1 Gene
The HWP1 gene was amplified to discriminate Candida albicans complex, including C. albicans, C. africana
and C. dubliniensis (16). The PCR with paired primers
HWP1-F (5′ - GCTACCACTTCAGAATCATCATC-3′ ) and HWP1-R
(5′ - GCACCTTCAGTCGTAGAGACG-3′ ) was done using the following condition: 15 minutes at 95°C; 35 cycles of 30 seconds at 95°C, annealing for 30 seconds at 60°C, 20 seconds
at 72°C, and a final extension of 1 minutes at 72°C. The bands
were confirmed by the determination of the band size.
3.3. In vitro Antifungal Susceptibilities Testing
The conidial suspensions were obtained from Aspergillus spp., dematiaceous spp., and Fusarium spp. after
seven days of growth on potato dextrose agar (Merck, Germany) and SDA at 35°C, respectively. For yeast spp., conidial suspensions were prepared from the 24-hour fresh culture at 35°C. Obtained suspensions were counted by hemocytometer, and the cell density was adjusted to 0.4 - 2.5
× 104 CFU/mL, and 0.5 × 103 to 2.5 × 103 for filamentous
and yeast isolates, respectively. The antifungal susceptibility testing to itraconazole, voriconazole, and posaconazole
was performed according to the CLSI-M38-3rd ed. (for filamentous fungi) (17) and CLSI-M60 (for yeast) guidelines
(18). All antifungal agents were purchased from the Sigma
Aldrich (USA). Candida parapsilosis ATCC 22019 was used as
a quality control strain. All tests were performed in duplicate.
4.2. Molecular Identification Results
The results of the PCR-based sequencing showed that
Fusarium solani (15) was the most frequent ocular species,
followed by A. flavus (10), A. fumigatus (7), C. albicans (5),
Curvularia spp. (4), F. oxysporum (3), A. niger (2), A. terreus
(1), F. Keratoplasticum (1), F. fujikuroi (1), F. falciforme (1), F.
proliferatum (1), F. verticillioides (1), R. rubra (1), and A. alternata (1). The ITS sequences of A section flavi indicated that
one isolate was A. orezae and others were A. flavus. All A.
section fumigati belonged to A. fumigatus. Some sequences
were deposited in the GenBank under accession numbers
of MT160346, MT160090, MT160089, MT160088, MT161477,
XI2321525, XI2321546, and XI2321600. All C. albicans complex strains revealed a 100% similarity to C. albicans (~ 900
bp) (Figure 1).
4.3. Analyses of Antifungal Susceptibility Pattern
The MIC range was calculated for all tested isolates (Table 1). Voriconazole and posaconazole showed excellent
activity in all tested isolates, however, some Fusarium, Aspergillus, and Curvularia strains showed MICs ≥ 2 µg/mL.
The itraconazole showed different results in all species
with high MICs (≥ 16 µg/mL).
3.4. Statistical Analyses
The statistical analysis was performed by Statistical
Package for Social Sciences (SPSS) V. 22.0 for Windows (SPSS
Inc., Chicago, IL, USA). The minimum inhibitory concentration (MIC50 /MIC90 ) and frequency of studied isolates were
determined using the t-test.
4. Results
4.1. Direct Examination and Morphological Analysis
Considering the morphology-based approaches
(macroscopic and microscopic features) Fusarium spp.
(22) were identified as the most common etiologic agents,
followed by Aspergillus flavus (10), A. fumigatus (7), C. albicans complex (5), dematiaceous spp. (4), A. niger (2), A.
terreus (1), Rhodotorula rubra (1), and Alternaria alternata (1).
Jundishapur J Microbiol. 2020; 13(3):e99922.
Figure 1. Agarose gel electrophoresis of HWP1 gene amplification of Candida albicans
(~ 900 bp); M, marker 50 bp.
3
Soleimani M et al.
Table 1. Comparison of MIC50 and MIC90 , and the MIC Range for all Tested Isolates
Fungal Species
Fusarium solani (n = 15)
F. oxysporum (n = 3)
F. keratoplasticum (n = 1)
Fusarium spp.
F. fujikuroi (n = 1)
F. falciforme (n = 1)
F. proliferatum (n = 1)
MIC50 /MIC90
Aspergillus flavus (n = 10)
A. fumigatus (n = 7)
Aspergillus spp.
A. niger (n = 2)
A.oryzae (n = 1)
A. terreus (n = 1)
MIC50 /MIC90
Yeast
Dematiaceous spp.
Candida albicans (n=5)
Curvularia spp.
Antifungal Agent
MIC Range (µg/mL)
ITZ
0.5 - 16
VCZ
0.0313 - 0.5
PCZ
0.0313 - 0.128
ITZ
16
VCZ
0.128
PCZ
0.0313
ITZ
0.128
VCZ
0.0313
PCZ
16
ITZ
0.128
VCZ
0.128
PCZ
0.0313
ITZ
0.0313
VCZ
0.0313
PCZ
0.0313
ITZ
0.0313
VCZ
0.0313
PCZ
0.0313
ITZ
1/2
VCZ
0.0313/0.5
PCZ
0.0313
ITZ
0.5 - 8
VCZ
0.125 - 2
PCZ
0.125
ITZ
2
VCZ
0.5
PCZ
0.5
ITZ
1
VCZ
1
PCZ
0.5
ITZ
0.125
VCZ
0.5
PCZ
0.125
ITZ
1
VCZ
0.125
PCZ
0.5
ITZ
0.5/2
VCZ
0.125/1
PCZ
0.125/0.5
ITZ
0.125 - 4
VCZ
0.125 - 0.5
PCZ
0.5
ITZ
0.5 - 8
VCZ
0.125 - 4
PCZ
0.125
Abbreviations: ITZ, itraconazole; MIC, minimal inhibitory concentration; PCZ, posaconazole; VCZ, voriconazole.
5. Discussion
Fungal ocular infection is common in Iran, and in the
present study, Fusarium spp. and Aspergillus spp. were responsible for 82% of the infection cases. Due to lack of
molecular identification of fungal ocular infection, the isolates have not been reported previously from Iran. In the
present study, 15 isolates out of 22 Fusarium spp. isolates
4
were known as F. solani. However, TEFα sequence data at
NCBI BLAST showed that 3 isolates were F. oxysporum, and 5
isolates were identified as F. Keratoplasticum (1), F. fujikuroi
(1), F. falciforme (1), F. proliferatum (1), and F. verticillioides (1),
which were reported for the first time as a causative agent
of fungal ocular infection in Iran. This finding is consistent
with that of previous research conducted in Iran that reported Fusarium spp. responsible for most cases of fungal
Jundishapur J Microbiol. 2020; 13(3):e99922.
Soleimani M et al.
keratitis, followed by Aspergillus spp. and Mucor spp. (13, 19,
20).
Also, in recent studies using ITS sequencing, most isolates were known as F. solani complex (2, 21-24). In a study
by Haghani et al., Aspergillus spp., especially A. flavus was
reported as the most prevalent etiologic agents (23). ITS sequencing at NCBI BLAST revealed A. flavus (10) and A. fumigatus (7) as the most common etiologic agents of Aspergillus
keratitis. In a study conducted in India, A. flavus was reported as the most important agent of Aspergillus keratitis
(2). In contrast, A. niger has previously been reported as the
most common cause of fungal ocular infection in Iran (21).
In the present study, A. orezae and A. terreus were isolated
from fungal ocular infection for the first time. In this research, four Curvularia spp., one R. rubra, and one A. alternata species were also identified. In a study from Iran one
Curvularia spp. was determined (23). Furthermore, C. albicans strains were responsible for fungal ocular infection.
Itraconazole and synthetic dioxalin and triazole have
resulted in a good clinical outcome to treat keratitis caused
by Aspergillus and Curvularia, even in severe and non-severe
forms of Fusarium keratitis (25). The results of antifungal
susceptibility to Fusarium spp. are conflicting, and a poor
in vitro activity of itraconazole has been reported (2, 25).
It has shown that the susceptibility to antifungal agents
is species-related, and the highest MICs of F. solani has
been reported (2, 26). In line with previous studies, our
results showed that itraconazole has a poor in vitro activity against Fusarium spp. (Table 1). Most F. solani strains
had the highest MIC to itraconazole (MIC ≥ 16). The emergence of less common fungal species resistant to itraconazole has been considered as a global concern. In this research, F. oxysporum and F. Keratoplasticum showed MIC ≥
16, which confirms that voriconazole and posaconazole are
better choices to treat corneal infection (P < 0.005).
Aspergillus spp. responded well to all agents. Only two
A. flavus isolates were resistant to itraconazole. A good in
vitro activity of voriconazole and itraconazole against Aspergillus spp. has been reported (25, 27). In the present
study, all C. albicans isolates were susceptible to azoles and
exhibited MIC values of 0.125 - 4, 0.625 - 0.05, and 0.125 0.5 µg/mL for itraconazole, voriconazole, and posaconazole, respectively. The lowest MIC values of itraconazole
against Candida spp. isolated from corneal infection has
been recorded previously (26). This finding confirms the
therapeutic potential of itraconazole, voriconazole and
posaconazole to treat keratitis caused by C. albicans (P <
0.005). The results of the susceptibility tests showed that
posaconazole was the most active drug against Curvularia
spp. Itraconazole and voriconazole generally showed good
activity; however, one isolate showed high MICs (8 and
4 µg/mL) to itraconazole and voriconazole, respectively.
Jundishapur J Microbiol. 2020; 13(3):e99922.
The highest MICs of itraconazole and voriconazole against
some species of Curvularia, including C. aeria, C. borreriae,
C. protuberata, and C. pseudorobusta has been reported (28).
5.1. Conclusions
Finally, in the present study, we tried to identify species
involved in fungal ocular infection using the molecular
methods, which highlighted the importance of precise
identification of species to choose an appropriate antifungal regime. On the other hand, our findings showed that
antifungal susceptibility test is effective to reliably predict
the in vivo response to therapy in infections; however, in
fungal ocular infection cases, the penetration of antifungals may contribute to predict the outcome.
Footnotes
Authors’ Contribution: Study concept and design: Mohammad Soleimani, Azam Fattahi; acquisition of data:
Ensieh Lotfali, Zahra Salehi, Zohreh Abedinifar; analysis
and interpretation of data: Ensieh Lotfali, Reza Ghasemi,
Zeynab Yassin; drafting of the manuscript: Azam Fattahi,
Fardin Ahmadkhani; administrative, technical support:
Azam Fattahi, Ebrahim Kouhsari, and Shiva Mirkalantari.
Conflict of Interests: The authors declared no conflict of
interest.
Ethical Approval: All procedures performed in studies involving human participants were in accordance with
the ethical standards of the Institutional and/or National
Research Committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Funding/Support: This study was financially supported
by the Iran University of Medical Sciences (grant no. 96-0430-32391).
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