Morphological Changes in Fungal Elements and Antifungal Drug Susceptibility in an Ex Vivo Model of Fungal Keratitis

Tomoko Miyakubo; Takashi Tamura; Aya Komori; Takane Ueda; Noriaki Ishioka; Daisuke Todokoro; Koichi Makimura

doi:10.1167/iovs.67.10.13

. 2026 Aug 5;67(10):13. doi: 10.1167/iovs.67.10.13

Morphological Changes in Fungal Elements and Antifungal Drug Susceptibility in an Ex Vivo Model of Fungal Keratitis

Tomoko Miyakubo ^1,^2,^✉, Takashi Tamura ³, Aya Komori ³, Takane Ueda ³, Noriaki Ishioka ³, Daisuke Todokoro ², Koichi Makimura ^1,³

PMCID: PMC13450652 PMID: 42554406

Abstract

Purpose

Fungal keratitis is a serious eye infection that can cause irreversible vision loss. One of the causative fungal pathogens, Fusarium solani (Neocosmospora solani), shows limited susceptibility to antifungal drugs. Furthermore, differences between in vitro and clinical outcomes are encountered in some cases, complicating treatment. In this study, we compared in vitro and ex vivo antifungal susceptibility using a porcine corneal model of F. solani keratitis.

Methods

An ex vivo fungal keratitis model was developed using porcine corneas, which were inoculated with F. solani and assigned to four treatment groups: natamycin (NAT), voriconazole (VRCZ), luliconazole (LLCZ), or saline (uninfected control). We compared the antifungal efficacy of each drug on day 2 to day 4 of fungal development. DNA was extracted from all corneas and used in real-time polymerase chain reaction to quantitatively assess fungal growth. Fungal morphology was examined by digital microscopy.

Results

All antifungal treatment groups demonstrated antifungal activity. The NAT-treated group showed clear inhibition of fungal growth as well as fungal structural damage. The VRCZ-treated group exhibited growth inhibition; however, fungal growth persisted even after additional drug administration. The LLCZ-treated group not only suppressed fungal growth but also caused structural disruption of fungal cells after additional administration, suggesting a strong antifungal effect.

Conclusions

We successfully established an ex vivo Fusarium keratitis model using porcine corneas, which enabled us to evaluate antifungal susceptibility by quantifying fungal growth and observing fungal morphological changes.

Keywords: fungal keratitis, ex vivo model, Fusarium solani, antifungal drug sensitivity, real-time PCR

Fungal keratitis is a corneal infection caused by filamentous fungi or yeasts. The annual global incidence is estimated to be approximately 1.05 million cases.¹ This disease occurs predominantly in tropical and subtropical regions of Asia and Africa, which account for most of the cases worldwide.² Over 95% of cases are caused by the filamentous fungi Fusarium spp. (particularly F. solani; Neocosmospora solani), Aspergillus spp., and Candida spp. Globally, fungal keratitis is responsible for vision loss in an estimated one million individuals each year, and enucleation is required in approximately 8%–11% of cases, corresponding to 95,000–115,800 eyes lost annually.¹ The inherently slow growth rate of fungi means that the diagnosis and initiation of treatment for fungal keratitis are often delayed, resulting in irreversible visual impairment, corneal perforation, or even loss of the eye.

Natamycin (NAT) is presently the only topical ophthalmic antifungal agent approved by the U.S. Food and Drug Administration, and treatment options for fungal keratitis continue to be more limited than those for bacterial keratitis. Other antifungals, such as voriconazole (VRCZ), are also used as topical treatment but are not widely commercially available. Luliconazole (LLCZ) is an imidazole antifungal agent commonly used to treat dermatophytosis and has potent activity against a wide range of filamentous fungi³^,⁴; therefore it is considered a promising treatment for Fusarium keratitis. Fusarium spp. are often multidrug-resistant, with 85% of clinically isolated Fusarium spp. reported to be resistant to VRCZ in the USA,⁵^,⁶ and many are also resistant to echinocandins and polyenes.⁷^–⁹ Moreover, inconsistencies between the results of in vitro antifungal susceptibility testing and clinical outcomes have been reported,¹⁰ which complicate treatment.

We have investigated these problems in an ex vivo model of keratitis. This model uses corneas excised from enucleated eyes and allows direct observation of corneal tissue invasion by the pathogen and associated structural changes. Although this model does not recapitulate the in vivo immune response, it is widely used to evaluate therapeutic strategies,¹¹^,¹² elucidate mechanisms of infection,¹³ assess whether the pathogen can use the cornea as a favorable substrate, and serve as a screening step before in vivo animal studies. The purpose of the present study was to compare the in vitro antifungal susceptibility of F. solani with the efficacy of antifungal agents in an ex vivo porcine model of keratitis. This evaluation represent a novel approach because it combines morphological observation of the hyphal changes induced by each antifungal agent with quantitative assessment of the fungal burden.

Methods

Antifungal Agents

The antifungal agents used were NAT (Sigma-Aldrich, St. Louis, MO, USA), VRCZ (Fluka; Sigma-Aldrich), and LLCZ (Nihon Nohyaku Co., Ltd, Tokyo, Japan). All three antifungal agents were dissolved in dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and then diluted in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) to the following final concentrations: NAT 3200 µg/mL; VRCZ 1600 µg/mL; and LLCZ 6 µg/mL.

Fungal Strain

A clinical isolate from fungal keratitis, F. solani (LSEM 4652), was obtained from the culture collection held by the Laboratory of Systematics and Environmental Mycology at the Teikyo University Institute of Medical Mycology in Japan. This strain was used for antifungal susceptibility testing and the ex vivo model developed in this study. DNA was extracted from the clinical isolate and subjected to amplification of two loci by PCR. The partial elongation factor-1 alpha (EF1-α) gene was amplified by PCR with the primer pair HS392/HS393,¹⁴ and the nuclear ribosomal internal transcribed spacer (ITS) region (ITS1, 5.8S and ITS2) was amplified using the primer pair ITS5/ITS4.¹⁵ The PCR products of the EF1-α and ITS regions were sequenced using the Sanger method, and the resulting sequences were compared with those in the MycoBank database. The sequences showed 100% identity to MN652893 and 100% identity to OR150403, both of which are members of the F. solani species complex (FSSC). Their phylogenetic placement was consistent with FSSC5 (F. solani sensu stricto). Accordingly, the isolate was identified as FSSC5. The clinical isolate was stored at −80°C until use.

In Vitro Study of Antifungal Susceptibility

Drug susceptibility testing was performed according to the Clinical and Laboratory Standards Institute M38, third edition, using the microbroth dilution protocol.¹⁶ Fungal cultures for antifungal activity testing were grown in RPMI 1640 medium supplemented with MOPS [3-(N-morpholino) propane sulfonic acid; Fujifilm Wako Pure Chemical Corporation] at 35°C for 48 hours.

The inoculum suspensions of F. solani conidia were adjusted to a McFarland standard of 0.5–1.0 corresponding to approximately 1 × 10⁶ conidia/mL. Serial two-fold dilutions of antifungal agents were prepared in 96-well microtiter plates containing RPMI 1640 medium supplemented with MOPS, with concentration ranges of 0.03–16 µg/mL for NAT, 0.015–8 µg/mL for VRCZ, and 0.00006–0.03 µg/mL for LLCZ. The plates were inoculated with the fungal culture and incubated at 35°C for 48 hours. Growth controls (medium with inoculum but no antifungal agent) were included. The MIC was defined as the minimum inhibitory concentration of antifungal agent required to inhibit growth by 50% compared with the control.

Ex Vivo Study

Preparation of the Inoculum

F. solani was cultured on potato dextrose agar (Kanto Chemical Co., Inc., Tokyo, Japan) at 28°C for five days. Microconidia were harvested from the colony surface using a disposable loop, then suspended in 2 mL of sterile saline solution, and filtered. The resulting suspension was transferred to a round-bottom tube and vortexed thoroughly. Spore concentrations were determined using a hemocytometer and adjusted to 8.0 × 10⁶ spores/mL.

Corneal Epithelial Detachment and Topical Inoculation

The ex vivo model was established using previously described methods,¹⁷^–¹⁹ with some modifications. Cadaveric porcine eyes (Tokyo Shibaura Zouki Co., Ltd, Tokyo, Japan) were shipped on ice and received within 12 hours of enucleation. Before topical inoculation, each eye was disinfected with 1.25% povidone–iodine and rinsed with sterile saline solution. The center of each cornea was marked using a 3 mm circular trephine, and the anterior half of the stroma was dissected using a surgical blade. The corneas were then excised from the globes and incubated at 4°C for three hours in sterile saline solution supplemented with gentamicin (50 µg/mL; Fujifilm Wako Pure Chemical Corporation), tetracycline (50 µg/mL; Fujifilm Wako Pure Chemical Corporation). Micafungin (10 µg/mL; Funguard, Astellas Pharma Inc., Tokyo, Japan) was also included for prophylaxis against yeast contamination, given that Fusarium spp. generally exhibit intrinsic resistance to this agent. After equilibration to room temperature, 10 µL of F. solani conidial suspension (1 × 10⁵ spores/10 µL) was applied to the de-epithelialized corneal surface for inoculation. The corneal buttons were placed with the epithelial side facing downward in a spot plate, and culture medium-agarose (0.5% w/v) solution was pipetted into the endothelial side of the cornea. The solution was allowed to solidify, and the corneal buttons were inverted so that the epithelial side was facing upward and positioned in the center of sterile culture dishes containing culture medium. The culture medium consisted of RPMI 1640 supplemented with penicillin (100 U/mL; Fujifilm Wako Pure Chemical Corporation), streptomycin (100 µg/mL; Fujifilm Wako Pure Chemical Corporation), gentamicin (50 µg/mL), micafungin (1 µg/mL), and HEPES (15 mM; Thermo Fisher Scientific). The dishes were incubated at 35°C for four days, with replacement of medium every 24 hours. Figure 1 provides an overview of how the ex vivo model was prepared.

Figure 1. — Schematic representation of the ex vivo model in cross section **(A)** and a top-view photograph **(B)**.

Antifungal Treatment

For each experimental condition, samples were divided into three groups: (1) a noninfected group without fungal inoculation or antifungal treatment, (2) an untreated group with fungal inoculation but no treatment, and (3) a treatment group receiving NAT, VRCZ, or LLCZ on days 2 and 3. Based on the treatment regimen for severe fungal keratitis,²⁰^,²¹ antifungal agents were administered five times daily (every two hours from 9:00 AM to 5:00 PM) on days 2 and 3 of incubation, with 50 µL per instillation. In the noninfected and untreated groups, 50 µL of sterile saline were applied instead of antifungal agents. Each antifungal agent (NAT, VRCZ, or LLCZ) was evaluated in an independent experiment that included a corresponding noninfected and untreated group. To evaluate changes in fungal burden, samples were collected on days 2, 3, and 4. Before sample collection, corneal surfaces were examined using a digital microscope (VHX-6000; Keyence, Osaka, Japan). Each experiment was performed in triplicate. The experimental schedule is summarized in Table 1.

Table 1.

Experimental Schedule

		Sample Collection
Group	Day 1	Day 2	Day 3	Day 4
NAT-treated group
Noninfected control (non-inoculated)	—	—	—	—
Untreated control (inoculated)	Inoculation	—	—	—
Antifungal treatment (inoculated)	Inoculation	NAT treatment	NAT treatment	—
VRCZ-treated group
Noninfected control (non-inoculated)	—	—	—	—
Untreated control (inoculated)	Inoculation	—	—	—
Antifungal treatment (inoculated)	Inoculation	VRCZ treatment	VRCZ treatment	—
LLCZ-treated group
Noninfected control (non-inoculated)	—	—	—	—
Untreated control (inoculated)	Inoculation	—	—	—
Antifungal treatment (inoculated)	Inoculation	LLCZ treatment	LLCZ treatment	—

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DNA Extraction

Fungal DNA was extracted from corneal samples collected on days 2, 3, and 4 from each group using the QIAamp Fast DNA Tissue Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions. In view of the large sample size, DNA extraction was performed after bisecting each cornea.

Measurement of Total Fungal DNA Using Real-Time Quantitative PCR

The copy number of F. solani DNA extracted from the samples was quantified by real-time quantitative PCR (qPCR). This assay used the F. solani–specific primers and probe described by Yang et al.,²² which were synthesized by BEX Co., Ltd (Tokyo, Japan). qPCR was performed in a 20 µL reaction mixture containing 10 µL of Probe qPCR Mix (Takara Bio Inc., Shiga, Japan), 10 µM each of forward and reverse primers, 10 µM of probe, 0.2 µL of ROX Reference Dye II (Takara Bio Inc.), 6.2 µL of Milli-Q water (Millipore, Burlington, MA, USA), and 2 µL of DNA template. Amplification was conducted using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific) under the following conditions: 94°C for 30 seconds, followed by 35 cycles of denaturation at 95°C for 30 seconds, and combined annealing/extension at 64°C for one minute.

The limit of quantification was defined by the lowest standard concentration (Ct ∼ 27). Amplification curves detected ≥3 cycles after this point (Ct > 30) were regarded as beyond the quantifiable range and treated as negative. The statistical analysis was performed with two-way ANOVA followed by Sidak's multiple comparisons test using GraphPad Prism ver.11 (GraphPad Software Inc., Boston, MA, USA).

Assessment of Viability

To evaluate fungal viability, additional ex vivo experiments were performed under the same drug conditions as described above with incubation at 35°C for four days. In addition to the original experimental groups, two vehicle control groups were included: V-NAT (with the same DMSO concentration as used in the NAT-treated group; DMSO 4.69%) and V-VL (with the same DMSO concentration as used in the VRCZ and LLCZ-treated groups; DMSO 2.34%). All antifungal treatment groups and vehicle control groups were evaluated in parallel within the same experiment using shared noninfected and untreated control groups. Each experimental group included six samples.

On day 4, the corneal samples were homogenized. One half of each sample was subjected to qPCR analysis, and the remaining half was serially diluted, plated onto agar medium, and incubated for colony enumeration.

Statistical analyses were performed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Overall differences among groups were assessed using ANOVA. Pairwise comparisons were conducted between the untreated group and each vehicle control group and between each treatment group and its corresponding vehicle control group using Welch's t-test with Holm's correction for multiple comparisons. A P-value < 0.05 was considered statistically significant.

Measurement of Hyphal Diameter

For improved visualization of hyphae, corneas cultured under the same conditions as described above were washed with saline solution, then covered with a 1:1 mixture of 10% KOH and Calcofluor White solution for 10 minutes, followed by three washes with saline solution. The stained corneas were examined using a fluorescence microscope (DM2500; Leica Microsystems, Wetzlar, Germany), and the diameters of hyphae on the corneal surface were measured. Hyphal diameter was measured at the widest point of the apical segment, defined as the region from the hyphal tip to the first septum, under magnification ×400. Hyphae showing obvious spherical swelling resembling chlamydospore-like structures were excluded. Ten hyphae were measured per experimental group, and the mean diameter was used for analysis.

The statistical analysis was performed using the Mann–Whitney U test with Holm's correction for multiple comparisons in Microsoft Excel. Comparisons were made between the untreated group and each vehicle control group and between each treatment group and its corresponding vehicle control. A P-value ≤ 0.05 was considered significant.

Histological Assessment

Fungal invasion was evaluated by subjecting corneal samples collected from the untreated group on day 2 of culture to histological analysis using periodic acid–Schiff staining.

Results

Fungal Strain

A giant colony of F. solani (LSEM4652) was cultured on potato dextrose agar at 28°C for five days (Fig. 2). The colony surface was white and cotton-like in texture with a yellow-to-whitish appearance on the reverse side.

Figure 2. — A giant colony of F. *solani* (LSEM4652) was cultured on potato dextrose agar at 28°C for five days. The colony had a white cotton-like surface **(A)** and was yellowish-white on the reverse side **(B)**.

In Vitro Study of Antifungal Susceptibility

The MICs of F. solani (LSEM4652) for NAT, VRCZ, and LLCZ were 8 µg/mL, 8 µg/mL, and 0.03 µg/mL, respectively. Antifungal solutions for the ex vivo experiments were prepared at the following concentrations: NAT, 150 µg/mL (0.015% w/v); VRCZ, 30 µg/mL (0.003% w/v); and LLCZ, 30 µg/mL (0.003% w/v). Considering the absence of drug clearance in the ex vivo system and the need to avoid overestimation of efficacy through drug accumulation, concentrations substantially below those used clinically were selected. Specifically, NAT was used at 150 µg/mL (vs 50,000 µg/mL in the 5% clinical formulation), VRCZ at 30 µg/mL (vs 10,000 µg/mL in a 0.1%–1% formulation), and LLCZ at 30 µg/mL.

Ex Vivo Study

Lesion sizes on the corneal surface were smaller in all antifungal-treated groups than in the untreated group (Fig. 3). In the untreated group, the fungal mass further elongated and grew from Day 3 to Day 4, showing the yellowish-white color commonly observed in Fusarium. In contrast, in the treated groups, fungal growth was suppressed, and therefore distinct fungal mass formation with such a yellowish-white color did not occur, resulting in a different color appearance from that in the untreated group. Microscopic observations are shown in Figure 4. In the NAT-treated group, digital microscopy revealed a tendency toward swelling of hyphal tips. In the VRCZ-treated group, there were fewer aerial hyphae on the corneal surface; however, the hyphal structures remained intact and visible. In the LLCZ-treated group, digital microscopy showed narrowing of hyphal diameters and blurred hyphal contours.

Figure 3. — Microscopic images of the corneal samples on day 4 of culture. **(A)** Noninfected control. *Black arrows* show the corneal margin and *white arrows* show the de-epithelialized area. **(B)** Untreated control. **(C)** NAT-treated group. **(D)** VRCZ-treated group. **(E)** LLCZ-treated group.

Figure 4. — Microscopic images of corneal samples on day 4 of culture. **(A)** Noninfected control (×100 magnification). **(B)** Untreated control (×500). **(C)** NAT-treated group (500×). **(D)** VRCZ-treated group (×500). (E) LLCZ-treated group (×500).

The total amounts of F. solani DNA obtained are shown as the mean ± SD in Table 2; the statistical significance of the changes over four days are indicated in Figure 5. For each experimental group, the data were normalized to the day 4 value in the untreated group (defined as 100%). The absolute values are provided in Supplementary Figure S1. In all experimental groups, values in the noninfected group were below the limit of detection. One replicate showed late amplification (Ct = 32), which was not reproducible and therefore considered nonspecific. Although some variability was observed in the untreated group, the total amounts of DNA on day 4 were significantly smaller in all antifungal-treated groups than in the corresponding untreated groups (NAT, P = 0.0121; VRCZ, P = 0.0029; LLCZ, P = 0.0052).

Table 2.

Total Amount of DNA of Fusarium solani in Each Experimental Group

Group	Day 2	Day 3	Day 4
NAT-treated group
Noninfected control	BLD	BLD	BLD
Untreated control	3.47 × 10⁷ ± 1.25 × 10⁷	9.20 × 10⁷ ± 2.51 × 10⁷	1.74 × 10⁸ ± 7.35 × 10⁷
Antifungal treatment	3.41 × 10⁷ ± 1.11 × 10⁷	6.00 × 10⁷ ± 1.39 × 10⁷	6.05 × 10⁷ ± 2.48 × 10⁷
VRCZ-treated group
Noninfected control	BLD	BLD	BLD
Untreated control	6.07 × 10⁷ ± 6.52 × 10⁶	6.58 × 10⁷ ± 1.05 × 10⁷	1.19 × 10⁸ ± 7.91 × 10⁶
Antifungal treatment	5.76 × 10⁷ ± 6.99 × 10⁶	3.77 × 10⁷ ± 5.60 × 10⁶	8.29 × 10⁷ ± 1.60 × 10⁷
LLCZ-treated group
Noninfected control	BLD	BLD	BLD
Untreated control	4.83 × 10⁷ ± 1.02 × 10⁷	1.66 × 10⁸ ± 4.63 × 10⁷	2.30 × 10⁸ ± 5.51 × 10⁷
Antifungal treatment	5.01 × 10⁷ ± 8.10 × 10⁶	1.15 × 10⁸ ± 4.49 × 10⁷	8.94 × 10⁷ ± 3.60 × 10⁷

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BLD, below the limit of detection.

Figure 5. — Normalized total amount of F. *solani* DNA in each experimental group. **(A)** NAT-treated group, **(B)** VRCZ-treated group, and **(C)** LLCZ-treated group. The data were normalized to day 4 of the untreated group. The statistical analysis was performed using two-way analysis of variance followed by Sidak's multiple comparisons test. Comparisons between the untreated and treated groups were performed at each time point. Data are presented as the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 versus the untreated control groups on the same sampling day.

On day 2, before treatment, the amounts of DNA in the groups treated with NAT, VRCZ, or LLCZ were similar to those in the corresponding untreated groups (NAT, P > 0.9999; VRCZ, P= 0.9776; LLCZ, P > 0.9999). On day 3, the total amount of DNA was significantly lower in the VRCZ-treated group than in the untreated group (P = 0.0177); however, no significant difference was observed in the NAT-treated and LLCZ-treated groups (NAT, P = 0.71; LLCZ, P = 0.4357).

The total amount of DNA increased in all the untreated groups over time. In the NAT experiment, there was a significant increase in the untreated group between days 2 and 4 (P = 0.0079) but not between days 2 and 3 (P = 0.7310), days 2 and 4 (P = 0.7215), or days 3 and 4 (P = 0.998). In the VRCZ experiment, the untreated group showed a significant increase between days 2 and 4 (P < 0.0001), a significant decrease between days 2 and 3 (P = 0.0259), and a significant increase between days 2 and 4 (P = 0.0074) and between days 3 and 4 (P = 0.0002). In the LLCZ experiment, the untreated control group showed a significant increase between days 2 and 3 (P = 0.0380) and between days 2 and 4 (P = 0.0039) but not between days 2 and 3, days 2 and 4, or days 3 and 4 (P = 0.2676, P = 0.5888, and P = 0.7853, respectively).

Assessment of Viability

The DNA quantified by qPCR and the CFU count on day 4 are shown for each group in Figure 6. CFU data were log₁₀-transformed for visualization. The qPCR analysis revealed no significant difference between the untreated group and each corresponding vehicle control group (untreated vs. V-NAT, P = 0.8184; untreated vs. V-VL, P = 0.9886). Although fungal DNA levels tended to be lower in the NAT-treated group than in the V-NAT group, the difference was not significant (P = 0.1706). Similarly, fungal DNA levels tended to be lower in the VRCZ-treated group than in the V-VL group (P = 0.3561), but the difference did not reach statistical significance. In contrast, fungal DNA levels were significantly lower in the LLCZ-treated group than in the V-VL group (P = 0.0111).

The CFU count was not significantly different between the untreated group and the corresponding vehicle control group but was significantly lower in all treatment groups than in the vehicle control groups (V-NAT vs. NAT, P < 0.001; V-VL vs. VRCZ, P = 0.001418; V-VL vs. LLCZ, P < 0.001). The CFU count decreased to below the detection limit in all samples in the NAT-treated group and in four of six samples in the LLCZ-treated group. In the VRCZ-treated group, the CFU count decreased to approximately 2.3% of that in the corresponding vehicle control group. A similar trend was observed in vitro, although the differences were not significant (Supplementary Fig. S2).

Hyphal Diameter

The mean hyphal diameter for each group obtained from the ex vivo experiments are shown in Figure 7. No significant difference in hyphal diameter was observed between the untreated group and the vehicle control groups (V-NAT and V-VL). A significant difference was observed between the V-NAT and NAT groups, with the NAT group exhibiting larger hyphal diameters (P < 0.05). No significant difference in hyphal diameter was found between the V-VL and VRCZ groups; however, hyphae were significantly thinner in the LLCZ group than in the V-VL group (P < 0.001). A similar trend was observed in the in vitro experiments, although the differences were not significant (Supplementary Figs. S3, S4).

Histological Assessment

In the untreated group, abundant hyphal growth was observed on the corneal surface on day 2 of culture, along with hyphae extending into the mid-stroma of the cornea (Fig. 8).

Figure 8. — Histological findings in the untreated group on day 2 of culture. Corneal sections from the untreated group on day 2 of culture were stained with periodic acid–Schiff. Abundant hyphal growth was observed on the corneal surface, along with hyphae extending into the mid-stroma (*arrows*).

Discussion

We have established an ex vivo porcine corneal model of F. solani keratitis that enables quantitative assessment of antifungal efficacy using qPCR. To demonstrate the applicability of this model, we evaluated three antifungal agents, including a polyene and two azoles, and observed that there were characteristic morphological changes associated with each agent.

NAT is a polyene macrolide antifungal agent and, along with VRCZ, is often used for topical treatment of fungal keratitis. More favorable outcomes are generally achieved with NAT than with VRCZ against Fusarium species, and NAT is recommended as a first-line treatment for filamentous keratitis, especially Fusarium keratitis.²³ NAT acts by specifically binding to ergosterol in the fungal cell membrane. Ergosterol stabilizes the membrane and participates in membrane fusion and fission during morphogenesis, including endocytosis, exocytosis, and vacuolar fusion. NAT inhibits these processes without compromising the integrity or permeability of the cell membrane.²⁴ Inhibition of transport protein complexes in the plasma membrane, which suppresses the uptake of various substrates, is considered one of the main mechanisms of the antifungal activity of NAT. As a result, membrane trafficking and component recycling at the hyphal tip are blocked, and vacuolar fragmentation impairs regulation of osmotic pressure, turgor, and cytoskeletal organization. These actions delay cell wall synthesis and supply to the membrane, disrupt polarized growth at hyphal tips, and ultimately suppress fungal growth. Our findings on digital microscopy and assessment of hyphal diameter suggested degenerative changes, including tip enlargement (spheroplast-like swelling) at sites of hyphal growth, which may reflect degenerative changes induced by exposure to NAT. In the NAT-treated group, the total amount of DNA showed no significant change between days 3 and 4, suggesting an inhibitory effect on growth. The finding that the CFU count fell below the limit of detection supports this interpretation.

VRCZ is a second-generation triazole antifungal agent and a synthetic derivative of fluconazole.²⁵ Multiple mechanisms have been associated with reduced susceptibility to azole in Fusarium spp., including alterations in CYP51 expression, efflux transporter activity, and formation of biofilm. Biofilm formation has been reported to increase the MIC of VRCZ in F. solani.²⁶^–³⁰ In our VRCZ-treated group, digital microscopy showed that the hyphal structure was maintained. Furthermore, the total amount of DNA decreased between day 2 and day 3 but increased again on day 4, suggesting partial regrowth of the fungus at the drug concentration administered. It has been reported that the MICs of VRCZ against Fusarium show a wide distribution with a mean of 4 µg/mL (range, 2–4).³¹ Previous studies have also suggested an increasing prevalence of azole-resistant strains.³²^,³³ The strain used in this study showed a relatively high MIC for VRCZ compared with previously reported ranges, suggesting reduced susceptibility to VRCZ. In both the ex vivo and in vitro CFU assays, viability was poorer in the VRCZ-treated groups than in the V-VL groups; however, viable fungi remained detectable. These results suggest that VRCZ may have acted predominantly in a growth-inhibitory (fungistatic) manner rather than exerting complete fungicidal activity against this strain under the experimental conditions used in this study.

LLCZ is an optically active imidazole antifungal that was developed in Japan as NND-502. This agent has been reported to have strong antifungal activity against Fusarium spp., with MIC₉₀ values of 0.005 µg/mL cited by Abastabar et al.³⁴ and 0.06 µg/mL by Todokoro et al.³⁵ The strain tested in this study also showed high susceptibility. The main mechanism of action of LLCZ is inhibition of ergosterol biosynthesis through inhibition of Cyp51. However, in vivo observations using scanning and transmission electron microscopy have suggested activity at both the plasma membrane and cell wall, depending on the local concentration.³⁶ In studies in which fungal cells have been treatment with LLCZ, scanning and transmission electron microscopy have revealed degenerative changes that indicate sites of action on the cell membrane, cell wall, and cytoplasm.³⁷^,³⁸ The narrowing of hyphal diameter and blurred outlines of hyphae seen in this study are consistent with these reports and similarly suggest pronounced structural degeneration of hyphae. Our findings are consistent with strong antifungal activity at the tested concentrations, in which the CFU count was below the detection limit in four of six samples, suggesting marked suppression of fungal viability. In the LLCZ-treated group, the total amount of DNA decreased further from day 3 to day 4, possibly reflecting breakdown or leakage of DNA in response to destruction of fungal cells. However, obvious hyphal disruption was not observed by in vitro microscopy.

In this study, all noninfected samples were below the detection limit, while the untreated groups and all antifungal-treated groups showed significant quantifiable levels, confirming the validity of the experimental system. In therapeutic studies using an ex vivo model of keratitis, fungal growth has mainly been evaluated by measuring lesion size on imaging or by CFU count in homogenized corneal samples. However, the CFU count does not accurately reflect the fungal burden in filamentous fungi. In this study, we directly quantified the DNA in F. solani in corneal tissues, which allowed clear numerical comparison and improved objectivity. Similar trends were observed in the CFU count, further supporting the validity of qPCR-based evaluation. However, the total amount of DNA in F. solani includes DNA derived from both viable and nonviable cells. Therefore, this measure reflects the overall fungal burden rather than the viable fungal count. The lack of an increase in DNA suggests growth suppression, but not necessarily fungicidal activity. Indeed, in all antifungal-treated groups, the total amount of DNA on day 4 was smaller than that in the untreated, indicating antifungal activity. Furthermore, the CFU counts on day 4 suggested fungicidal activity in the NAT-treated and LLCZ-treated groups and fungistatic activity in the VRCZ-treated group. These findings suggest that quantitative measurement of fungal growth in an ex vivo model of Fusarium keratitis can be used to evaluate antifungal susceptibility. However, each antifungal agent was evaluated in an independent experimental system with its own untreated control, and no direct comparisons between agents. Therefore the present study does not permit conclusions regarding the relative efficacy of the antifungal agents tested. Moreover, although these agents were administered five times daily, evaluations were performed only once day, precluding assessment of short-term pharmacodynamic behaviors such as transient suppression or regrowth between doses. Our results reflect cumulative drug effects rather than time-dependent sustained effects.

The methods of fungal inoculation in ex vivo models of infectious keratitis are similar to those used in in vivo models. Previously reported techniques include intrastromal injection,¹⁸ scarification of the corneal surface,¹⁷ and use of contact lenses.³⁹^,⁴⁰ In this study, we used inoculation with corneal surface scraping because of its suitability for observation of hyphal growth and created a central corneal groove into which we inoculated a suspension of fungal spores. This method reduced variation between samples caused by leakage from inoculate sites and achieved a more uniform inoculum dose. A fungal mass centered at the inoculation site was observed in all inoculated corneas. This approach minimized peripheral spread of the fungus and allowed assessment of hyphal morphology on the corneal surface. The validity of this model was supported by the finding that the total amount of DNA on day 2 was comparable across all antifungal-treated groups and the corresponding untreated groups, indicating consistent initial conditions and good reproducibility. Roberts et al.⁴¹ confirmed by microscopy that complete germination of F. keratoplatsticum occurred in vitro within 24 hours of incubation. They used a two-day incubation condition to model late-presenting fungal infection typical of clinical experience in an ex vivo setting and found that antifungal susceptibility decreased the longer treatment was delayed. Accordingly, in the present study, a two-day incubation period was included before exposure to antifungals. This approach enabled assessment of antifungal activity against mature mycelial forms, rather than the conidial stage typically used in standard MIC assays.

This study has several limitations. First, only one concentration of each antifungal agent was used and was significantly lower than the concentrations used in clinical practice. However, in vivo, drug levels are diluted by tears, so the concentrations tested in this study were not unrealistic. Moreover, even at these low concentrations, digital microscopy revealed characteristic morphological changes in response to exposure to each drug. Second, this ex vivo model does not recapitulate important in vivo factors, such as the immune response, inflammatory signaling, tear-film dynamics, and drug metabolism. However, our objective was to isolate and assess the direct effects of antifungal agents on fungal growth and morphology within a tissue-specific environment that cannot be reproduced in vitro, as well as the direct effects of antifungal agents that are difficult to isolate in vivo. Therefore we consider that this model can serve a translational role between in vitro and in vivo systems. Third, the study was conducted using a single strain isolated from a clinical specimen, which means that our findings cannot be generalized to all antifungal agents. The drug concentrations used were not measured within corneal tissue, so their relationship to actual tissue exposure remains unclear. Furthermore, only a single concentration was evaluated, and dose–response relationships were not investigated. Accordingly, while this study demonstrated the changes in morphology and fungal burden associated with antifungal exposure, it does not provide a comprehensive evaluation of pharmacodynamic properties. Fourth, interspecimen variability may have affected the quantitative assessment of fungal burden because corneal weight and surface area were not measured. Finally, variation in corneal thickness and nutrient diffusion could influence local drug concentrations and fungal invasion patterns. Addressing these factors in further studies will enhance the translational relevance of this model. In addition, the vehicle control groups contained up to 4.69% DMSO, which may have affected corneal viability and should be addressed in future research.

In conclusion, we have established an ex vivo porcine model of F. solani keratitis that enables quantitative and morphological assessment of antifungal efficacy, providing a bridge between in vitro susceptibility testing and clinical outcomes.

Supplementary Material

Supplement 1

iovs-67-10-13_s001.docx^{(548.7KB, docx)}

Acknowledgments

The authors thank Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

During the preparation of this work the authors used OpenAI's ChatGPT (GPT-5, accessed in October 2025) to improve readability and language. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Disclosure: T. Miyakubo, None; T. Tamura, None; A. Komori, None; T. Ueda, None; N. Ishioka, None; D. Todokoro, None; K. Makimura, SEREN Pharmaceuticals Inc. (F), TAKT Pharmaceuticals Inc. (F)

References

1. Brown L, Leck AK, Gichangi M, Burton MJ, Denning DW.. The global incidence and diagnosis of fungal keratitis. Lancet Infect Dis. 2021; 21(3): e49–e57. [DOI] [PubMed] [Google Scholar]
2. Kam KW, Rao SK, Young AL.. The diagnostic dilemma of fungal keratitis. Eye. 2023; 37: 386–387. [DOI] [PMC free article] [PubMed] [Google Scholar]
3. Khanna D, Bharti S.. Luliconazole for the treatment of fungal infections: an evidence-based review. Core Evid. 2014; 9: 113–124. [DOI] [PMC free article] [PubMed] [Google Scholar]
4. Niwano Y, Kuzuhara N, Kodama H, Yoshida M, Miyazaki T, Yamaguchi H.. In vitro and in vivo antidermatophyte activities of NND-502, a novel optically active imidazole antimycotic agent. Antimicrob Agents Chemother. 1998; 42: 967–970. [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Li D, Amburgey-Crovetti K, Applebach E, Steen TY, Calderone R.. The dual pathogen fusarium: diseases, incidence, azole resistance, and biofilms. J Fungi. 2025; 11: 294. [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Zhang R, Wiederhold N, Calderone R, Li D.. Biofilm Formation in Clinical Isolates of Fusarium. J Fungi. 2024; 10: 766. [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Al-Hatmi AM, Meis JF, de Hoog GS. Fusarium: molecular diversity and intrinsic drug resistance. PLoS Pathog. 2016; 12(4): e1005464. [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Alastruey-Izquierdo A, Cuenca-Estrella M, Monzon A, Mellado E, Rodriguez-Tudela JL.. Antifungal susceptibility profile of clinical Fusarium spp. isolates identified by molecular methods. J Antimicrob Chemother. 2008; 61: 805–809. [DOI] [PubMed] [Google Scholar]
9. Azor M, Gene J, Cano J, Guarro J. Universal in vitro antifungal resistance of genetic clades of the Fusarium solani species complex. Antimicrob Agents Chemother. 2007; 51: 1500–1503. [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Al-Hatmi AMS, Curfs-Breuker I, de Hoog GS, Meis JF, Verweij PE. Antifungal susceptibility testing of fusarium: a practical approach. J Fungi. 2017; 3(2): 19. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Bosman MA, Craig JP, Swift S, Dean SJ, Marasini S.. Therapeutic efficacy of ultraviolet C light on fungal keratitis-in vitro and ex vivo studies. Antibiotics. 2025; 14: 361. [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Marasini S, Dean SJ, Swift S, et al.. Preclinical confirmation of UVC efficacy in treating infectious keratitis. Ocul Surf. 2022; 25: 76–86. [DOI] [PubMed] [Google Scholar]
13. Urwin L, Okurowska K, Crowther G, et al.. Corneal infection models: tools to investigate the role of biofilms in bacterial keratitis. Cells. 2020; 9: 2450. [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Muraosa Y, Schreiber AZ, Trabasso P, et al.. Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC). Int J Med Microbiol. 2014; 304(3-4): 505–511. [DOI] [PubMed] [Google Scholar]
15. White TJ, Bruns T. D., Lee S. B., and Taylor J. W.. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications. San Diego: Academic Press; 1990. [Google Scholar]
16. Clinical and Laboratory Standards Institute: Reference method for broth dilution antifungal susceptibility testing of filamentous fungi (M38 3rd ed.). Wayne, PA: Clinical and Laboratory Standards Institute; 2017. [Google Scholar]
17. Hua X, Yuan X, Di Pietro A, Wilhelmus KR.. The molecular pathogenicity of Fusarium keratitis: a fungal transcriptional regulator promotes hyphal penetration of the cornea. Cornea. 2010; 29: 1440–1444. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Pinnock A, Shivshetty N, Roy S, et al.. Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis. Graefes Arch Clin Exp Ophthalmol. 2017; 255: 333–342. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Okurowska K, Roy S, Thokala P, et al.. Establishing a porcine ex vivo cornea model for studying drug treatments against bacterial keratitis. J Vis Exp. 2020;(159): e61156. [DOI] [PubMed] [Google Scholar]
20. Prajna NV, Mascarenhas J, Krishnan T, et al.. Comparison of natamycin and voriconazole for the treatment of fungal keratitis. Arch Ophthalmol. 2010; 128: 672–678. [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Sharma N, Sahay P, Maharana PK, et al.. Management algorithm for fungal keratitis: the TST (Topical, Systemic, and Targeted Therapy) Protocol. Cornea. 2019; 38: 141–145. [DOI] [PubMed] [Google Scholar]
22. Yang L, Zhou S, Nie D, et al.. Developing PCR-based novel molecular assays to quantitatively detect Fusarium solani in ginseng soil for assessing soil health in ginseng cultivation. Soil Science and Environment. 2023; 2(1): 2–7. [Google Scholar]
23. Prajna NV, Krishnan T, Mascarenhas J, et al.. The mycotic ulcer treatment trial: a randomized trial comparing natamycin vs. voriconazole. JAMA Ophthalmol. 2013; 131: 422–429. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. te Welscher YM, ten Napel HH, Balague MM, et al.. Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane. J Biol Chem. 2008; 283: 6393–6401. [DOI] [PubMed] [Google Scholar]
25. Johnson LB, Kauffman CA.. Voriconazole: a new triazole antifungal agent. Clin Infect Dis. 2003; 36: 630–637. [DOI] [PubMed] [Google Scholar]
26. D'Agostino M, Lemmet T, Dufay C, et al.. Overinduction of CYP51A gene after exposure to azole antifungals provides a first clue to resistance mechanism in Fusarium solani species complex. Microb Drug Resist. 2018; 24: 768–773. [DOI] [PubMed] [Google Scholar]
27. Cordeiro RA, Portela FV, Pereira LM, et al.. Efflux pump inhibition controls growth and enhances antifungal susceptibility of Fusarium solani species complex. Future Microbiol. 2020; 15: 9–20. [DOI] [PubMed] [Google Scholar]
28. Sav H, Rafati H, Oz Y, et al.. Biofilm formation and resistance to fungicides in clinically relevant members of the fungal genus fusarium. J Fungi. 2018; 4(1): 16. [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Mukherjee PK, Chandra J, Yu C, Sun Y, Pearlman E, Ghannoum MA.. Characterization of fusarium keratitis outbreak isolates: contribution of biofilms to antimicrobial resistance and pathogenesis. Invest Ophthalmol Vis Sci. 2012; 53: 4450–4457. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Cordova-Alcantara IM, Venegas-Cortes DL, Martinez-Rivera MA, Perez NO, Rodriguez-Tovar AV.. Biofilm characterization of Fusarium solani keratitis isolate: increased resistance to antifungals and UV light. J Microbiol. 2019; 57: 485–497. [DOI] [PubMed] [Google Scholar]
31. Sun CQ, Lalitha P, Prajna NV, et al.. Association between in vitro susceptibility to natamycin and voriconazole and clinical outcomes in fungal keratitis. Ophthalmology. 2014; 121: 1495–1500. [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Prajna NV, Lalitha P, Rajaraman R, et al.. Changing azole resistance: a secondary analysis of the MUTT I Randomized Clinical Trial. JAMA Ophthalmol. 2016; 134: 693–696. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Prajna NV, Lalitha P, Krishnan T, et al.. Patterns of antifungal resistance in adult patients with fungal keratitis in South India: a post hoc analysis of 3 randomized clinical trials. JAMA Ophthalmol. 2022; 140: 179–184. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Abastabar M, Al-Hatmi AMS, Vafaei Moghaddam M, et al.. Potent activities of luliconazole, lanoconazole, and eight comparators against molecularly characterized Fusarium species. Antimicrob Agents Chemother. 2018; 62(5): 10–1128. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Todokoro D, Suzuki T, Tamura T, et al.. Efficacy of luliconazole against broad-range filamentous fungi including Fusarium solani species complex causing fungal keratitis. Cornea. 2019; 38: 238–242. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Wakumoto-Nakashima K, Yamada N, Morino S, Yamamoto O.. Novel in vivo observations on double acting points of luliconazole on Trichophyton rubrum: an ultrastructural study. Med Mycol. 2015; 53: 860–867. [DOI] [PubMed] [Google Scholar]
37. Nishiyama Y, Maeda M, Yamada T.. Effect of topical antifungal luliconazole on hyphal morphology of trichophyton mentagrophytes grown on in vitro onychomycosis model. Mycopathologia. 2022; 187(5-6): 491–496. [DOI] [PubMed] [Google Scholar]
38. Ehara Y, Yamada N, Horie T, Furuichi R, Yoshida Y, Yamamoto O.. Novel in vivo observations of luliconazole 5% nail solution for onychomycosis: an ultrastructural study. Med Mycol. 2025; 63(3): myaf016. [DOI] [PubMed] [Google Scholar]
39. Koch JM, Refojo MF, Hanninen LA, Leong FL, Kenyon KR.. Experimental Pseudomonas aeruginosa keratitis from extended wear of soft contact lenses. Arch Ophthalmol. 1990; 108: 1453–1459. [DOI] [PubMed] [Google Scholar]
40. Lawin-Brussel CA, Refojo MF, Leong FL, Hanninen L, Kenyon KR.. Time course of experimental Pseudomonas aeruginosa keratitis in contact lens overwear. Arch Ophthalmol. 1990; 108: 1012–1019. [DOI] [PubMed] [Google Scholar]
41. Roberts D, Salmon J, Cubeta MA, Gilger BC.. Phase-dependent differential in vitro and ex vivo susceptibility of Aspergillus flavus and Fusarium keratoplasticum to azole antifungals. J Fungi. 2023; 9: 966. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1

iovs-67-10-13_s001.docx^{(548.7KB, docx)}

[bib1] 1. Brown L, Leck AK, Gichangi M, Burton MJ, Denning DW.. The global incidence and diagnosis of fungal keratitis. Lancet Infect Dis. 2021; 21(3): e49–e57. [DOI] [PubMed] [Google Scholar]

[bib2] 2. Kam KW, Rao SK, Young AL.. The diagnostic dilemma of fungal keratitis. Eye. 2023; 37: 386–387. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib3] 3. Khanna D, Bharti S.. Luliconazole for the treatment of fungal infections: an evidence-based review. Core Evid. 2014; 9: 113–124. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4. Niwano Y, Kuzuhara N, Kodama H, Yoshida M, Miyazaki T, Yamaguchi H.. In vitro and in vivo antidermatophyte activities of NND-502, a novel optically active imidazole antimycotic agent. Antimicrob Agents Chemother. 1998; 42: 967–970. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5. Li D, Amburgey-Crovetti K, Applebach E, Steen TY, Calderone R.. The dual pathogen fusarium: diseases, incidence, azole resistance, and biofilms. J Fungi. 2025; 11: 294. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib6] 6. Zhang R, Wiederhold N, Calderone R, Li D.. Biofilm Formation in Clinical Isolates of Fusarium. J Fungi. 2024; 10: 766. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib7] 7. Al-Hatmi AM, Meis JF, de Hoog GS. Fusarium: molecular diversity and intrinsic drug resistance. PLoS Pathog. 2016; 12(4): e1005464. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8. Alastruey-Izquierdo A, Cuenca-Estrella M, Monzon A, Mellado E, Rodriguez-Tudela JL.. Antifungal susceptibility profile of clinical Fusarium spp. isolates identified by molecular methods. J Antimicrob Chemother. 2008; 61: 805–809. [DOI] [PubMed] [Google Scholar]

[bib9] 9. Azor M, Gene J, Cano J, Guarro J. Universal in vitro antifungal resistance of genetic clades of the Fusarium solani species complex. Antimicrob Agents Chemother. 2007; 51: 1500–1503. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10. Al-Hatmi AMS, Curfs-Breuker I, de Hoog GS, Meis JF, Verweij PE. Antifungal susceptibility testing of fusarium: a practical approach. J Fungi. 2017; 3(2): 19. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib11] 11. Bosman MA, Craig JP, Swift S, Dean SJ, Marasini S.. Therapeutic efficacy of ultraviolet C light on fungal keratitis-in vitro and ex vivo studies. Antibiotics. 2025; 14: 361. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib12] 12. Marasini S, Dean SJ, Swift S, et al.. Preclinical confirmation of UVC efficacy in treating infectious keratitis. Ocul Surf. 2022; 25: 76–86. [DOI] [PubMed] [Google Scholar]

[bib13] 13. Urwin L, Okurowska K, Crowther G, et al.. Corneal infection models: tools to investigate the role of biofilms in bacterial keratitis. Cells. 2020; 9: 2450. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib14] 14. Muraosa Y, Schreiber AZ, Trabasso P, et al.. Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC). Int J Med Microbiol. 2014; 304(3-4): 505–511. [DOI] [PubMed] [Google Scholar]

[bib15] 15. White TJ, Bruns T. D., Lee S. B., and Taylor J. W.. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications. San Diego: Academic Press; 1990. [Google Scholar]

[bib16] 16. Clinical and Laboratory Standards Institute: Reference method for broth dilution antifungal susceptibility testing of filamentous fungi (M38 3rd ed.). Wayne, PA: Clinical and Laboratory Standards Institute; 2017. [Google Scholar]

[bib17] 17. Hua X, Yuan X, Di Pietro A, Wilhelmus KR.. The molecular pathogenicity of Fusarium keratitis: a fungal transcriptional regulator promotes hyphal penetration of the cornea. Cornea. 2010; 29: 1440–1444. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18. Pinnock A, Shivshetty N, Roy S, et al.. Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis. Graefes Arch Clin Exp Ophthalmol. 2017; 255: 333–342. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib19] 19. Okurowska K, Roy S, Thokala P, et al.. Establishing a porcine ex vivo cornea model for studying drug treatments against bacterial keratitis. J Vis Exp. 2020;(159): e61156. [DOI] [PubMed] [Google Scholar]

[bib20] 20. Prajna NV, Mascarenhas J, Krishnan T, et al.. Comparison of natamycin and voriconazole for the treatment of fungal keratitis. Arch Ophthalmol. 2010; 128: 672–678. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib21] 21. Sharma N, Sahay P, Maharana PK, et al.. Management algorithm for fungal keratitis: the TST (Topical, Systemic, and Targeted Therapy) Protocol. Cornea. 2019; 38: 141–145. [DOI] [PubMed] [Google Scholar]

[bib22] 22. Yang L, Zhou S, Nie D, et al.. Developing PCR-based novel molecular assays to quantitatively detect Fusarium solani in ginseng soil for assessing soil health in ginseng cultivation. Soil Science and Environment. 2023; 2(1): 2–7. [Google Scholar]

[bib23] 23. Prajna NV, Krishnan T, Mascarenhas J, et al.. The mycotic ulcer treatment trial: a randomized trial comparing natamycin vs. voriconazole. JAMA Ophthalmol. 2013; 131: 422–429. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib24] 24. te Welscher YM, ten Napel HH, Balague MM, et al.. Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane. J Biol Chem. 2008; 283: 6393–6401. [DOI] [PubMed] [Google Scholar]

[bib25] 25. Johnson LB, Kauffman CA.. Voriconazole: a new triazole antifungal agent. Clin Infect Dis. 2003; 36: 630–637. [DOI] [PubMed] [Google Scholar]

[bib26] 26. D'Agostino M, Lemmet T, Dufay C, et al.. Overinduction of CYP51A gene after exposure to azole antifungals provides a first clue to resistance mechanism in Fusarium solani species complex. Microb Drug Resist. 2018; 24: 768–773. [DOI] [PubMed] [Google Scholar]

[bib27] 27. Cordeiro RA, Portela FV, Pereira LM, et al.. Efflux pump inhibition controls growth and enhances antifungal susceptibility of Fusarium solani species complex. Future Microbiol. 2020; 15: 9–20. [DOI] [PubMed] [Google Scholar]

[bib28] 28. Sav H, Rafati H, Oz Y, et al.. Biofilm formation and resistance to fungicides in clinically relevant members of the fungal genus fusarium. J Fungi. 2018; 4(1): 16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib29] 29. Mukherjee PK, Chandra J, Yu C, Sun Y, Pearlman E, Ghannoum MA.. Characterization of fusarium keratitis outbreak isolates: contribution of biofilms to antimicrobial resistance and pathogenesis. Invest Ophthalmol Vis Sci. 2012; 53: 4450–4457. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib30] 30. Cordova-Alcantara IM, Venegas-Cortes DL, Martinez-Rivera MA, Perez NO, Rodriguez-Tovar AV.. Biofilm characterization of Fusarium solani keratitis isolate: increased resistance to antifungals and UV light. J Microbiol. 2019; 57: 485–497. [DOI] [PubMed] [Google Scholar]

[bib31] 31. Sun CQ, Lalitha P, Prajna NV, et al.. Association between in vitro susceptibility to natamycin and voriconazole and clinical outcomes in fungal keratitis. Ophthalmology. 2014; 121: 1495–1500. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib32] 32. Prajna NV, Lalitha P, Rajaraman R, et al.. Changing azole resistance: a secondary analysis of the MUTT I Randomized Clinical Trial. JAMA Ophthalmol. 2016; 134: 693–696. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib33] 33. Prajna NV, Lalitha P, Krishnan T, et al.. Patterns of antifungal resistance in adult patients with fungal keratitis in South India: a post hoc analysis of 3 randomized clinical trials. JAMA Ophthalmol. 2022; 140: 179–184. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib34] 34. Abastabar M, Al-Hatmi AMS, Vafaei Moghaddam M, et al.. Potent activities of luliconazole, lanoconazole, and eight comparators against molecularly characterized Fusarium species. Antimicrob Agents Chemother. 2018; 62(5): 10–1128. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib35] 35. Todokoro D, Suzuki T, Tamura T, et al.. Efficacy of luliconazole against broad-range filamentous fungi including Fusarium solani species complex causing fungal keratitis. Cornea. 2019; 38: 238–242. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib36] 36. Wakumoto-Nakashima K, Yamada N, Morino S, Yamamoto O.. Novel in vivo observations on double acting points of luliconazole on Trichophyton rubrum: an ultrastructural study. Med Mycol. 2015; 53: 860–867. [DOI] [PubMed] [Google Scholar]

[bib37] 37. Nishiyama Y, Maeda M, Yamada T.. Effect of topical antifungal luliconazole on hyphal morphology of trichophyton mentagrophytes grown on in vitro onychomycosis model. Mycopathologia. 2022; 187(5-6): 491–496. [DOI] [PubMed] [Google Scholar]

[bib38] 38. Ehara Y, Yamada N, Horie T, Furuichi R, Yoshida Y, Yamamoto O.. Novel in vivo observations of luliconazole 5% nail solution for onychomycosis: an ultrastructural study. Med Mycol. 2025; 63(3): myaf016. [DOI] [PubMed] [Google Scholar]

[bib39] 39. Koch JM, Refojo MF, Hanninen LA, Leong FL, Kenyon KR.. Experimental Pseudomonas aeruginosa keratitis from extended wear of soft contact lenses. Arch Ophthalmol. 1990; 108: 1453–1459. [DOI] [PubMed] [Google Scholar]

[bib40] 40. Lawin-Brussel CA, Refojo MF, Leong FL, Hanninen L, Kenyon KR.. Time course of experimental Pseudomonas aeruginosa keratitis in contact lens overwear. Arch Ophthalmol. 1990; 108: 1012–1019. [DOI] [PubMed] [Google Scholar]

[bib41] 41. Roberts D, Salmon J, Cubeta MA, Gilger BC.. Phase-dependent differential in vitro and ex vivo susceptibility of Aspergillus flavus and Fusarium keratoplasticum to azole antifungals. J Fungi. 2023; 9: 966. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Morphological Changes in Fungal Elements and Antifungal Drug Susceptibility in an Ex Vivo Model of Fungal Keratitis

Tomoko Miyakubo

Takashi Tamura

Aya Komori

Takane Ueda

Noriaki Ishioka

Daisuke Todokoro

Koichi Makimura

Abstract

Purpose

Methods

Results

Conclusions

Methods

Antifungal Agents

Fungal Strain

In Vitro Study of Antifungal Susceptibility

Ex Vivo Study

Preparation of the Inoculum

Corneal Epithelial Detachment and Topical Inoculation

Figure 1.

Antifungal Treatment

Table 1.

DNA Extraction

Measurement of Total Fungal DNA Using Real-Time Quantitative PCR

Assessment of Viability

Measurement of Hyphal Diameter

Histological Assessment

Results

Fungal Strain

Figure 2.

In Vitro Study of Antifungal Susceptibility

Ex Vivo Study

Figure 3.

Figure 4.

Table 2.

Figure 5.

Assessment of Viability

Figure 6.

Hyphal Diameter

Figure 7.

Histological Assessment

Figure 8.

Discussion

Supplementary Material

Acknowledgments

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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