Although antifungal supplementation reduces the fungal load in the corneal storage medium, consensus is lacking on the influence of dosing and temperature. The study aims to evaluate the impact of eye bank warming protocol and timing of antifungal supplements on efficacy in Optisol-GS and tissue.
Methods
Corneoscleral rims contaminated with Candida albicans (C. albicans) were incubated in Optisol-GS, either without antifungal agents or with the addition of amphotericin B or voriconazole at various concentrations (2 ×, 5 ×, 10 ×, and 20 × MIC), at different time points, and under various preservation temperatures (2–8 °C versus 2 h-room temperature exposure). Antifungal efficacy was evaluated by counting viable yeast colonies cultured from Optisol-GS samples. Tissue sterility was determined through direct tissue culture and histological examination of the contaminated rims after a 14-day incubation period.
Results
Room temperature exposure did not increase colony growth at the same multiple MIC of antifungal agents. Although antifungal addition reduced C. albicans growth in a concentration-dependent manner, yeast growth was still observed in all Optisol-GS samples with a single supplementation after a 14-day incubation. Only groups with additional antifungal supplementation on either day 2 or day 6 showed a 99% or greater reduction of C. albicans growth in Optisol-GS samples and yielded negative results in direct tissue culture.
Conclusions
The eye bank warming protocol did not compromise antifungal efficacy. To sustain the required concentration and effectively reduce C. albicans growth in Optisol-GS and contaminated tissue, additional antifungal supplementation on either day 2 or day 6 was necessary during a 2-week preservation period.
Improving prevention of fungal infection after endothelial keratoplasty is crucial because of its potential devastating nature.
Antifungal supplementation is known to reduce fungal load in the corneal storage medium.
Currently consensus on antifungal dosing and intervals in storage medium is lacking.
What was learned from the study?
The warming protocol regulated by eye banks does not appear to compromise antifungal efficacy.
Additional antifungal supplementation may be needed to reduce yeast growth during 2-week storage.
Introduction
The rise in post-endothelial keratoplasty (EK) fungal infections parallels the increase in the use of the EK procedure [1]. Post-EK keratitis and endophthalmitis are more commonly associated with fungal etiologies compared to those in penetrating keratoplasty. Given the grave consequences of post-EK fungal infections, eye banks have proposed several preventive strategies. These include rigorous donor screening [2], minimizing warming and processing times [3], improving aseptic techniques [4], and incorporating antifungal drugs into the storage medium [5‐9]. Eye banks typically employ two corneal storage methods: organ culture and hypothermic storage. Studies have shown a higher incidence of fungal infection in corneas stored under hypothermic conditions compared to those in organ culture [10]. The lower fungal infection rate associated with organ culture may be attributed to the extended preservation time, allowing for thorough screening of both tissue and culture medium before tissue release, as well as the inclusion of amphotericin B in the organ culture medium [11, 12]. Nevertheless, there is ongoing debate regarding the necessity of adding antifungal agents to hypothermic storage media and culturing the media before releasing the tissue.
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The use of antifungal supplementation in the storage medium has been proposed as a safe and cost-effective measure for EK [13‐15]. However, the majority of previous studies contributing to our foundational understanding of antifungal efficacy in storage media relied on models involving storage medium inoculation [5‐9], a scenario less likely to occur in real-world settings. Therefore, our study aims to replicate fungal contamination in tissue during retrieval and assess antifungal efficacy within the context of standard eye banking protocol by employing a novel simulation model.
Methods
Experimental Design
The study received approval from the Institutional Research Ethics Board of National Taiwan University Hospital (IRB No. 201904036RIND). All procedures were performed in accordance with the tenets of the Declaration of Helsinki of 1964 and its later amendments. Tissues used in the study were residual tissue from penetrating keratoplasty and procured in line with WHO Guiding Principles on Human Cell, Tissue and Organ Transplantation. Figure 1 outlines the study design.
Fig. 1
Flowchart of study design
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The initial phase of the study assessed the impact of room temperature exposure on antifungal efficacy against C. albicans-contaminated corneoscleral rims over a 7-day incubation period. Antifungal agents were applied on day 0 at concentrations of 2 ×, 5 ×, 10 ×, and 20 × MIC. Two temperature regimens were employed: regimen A maintained 2–8 °C throughout, and regimen B included a 2-h exposure to room temperature on days 1, 2, and 7 to mimic eye bank processing before tissue release. The second phase evaluated the antifungal effectiveness with varying supplementation timings under the current practice of 2-week hypothermic storage. Antifungal supplements at 20 × MIC were administered on day 0 (group A), days 0 and 2 (group B), days 0 and 6 (group C), or day 6 only (group D).
Antifungal efficacy was determined by viable yeast colonies cultured from Optisol-GS samples. Tissue sterility was evaluated through the direct recovery of C. albicans from tissue and histological examination of the contaminated rims after a 14-day incubation.
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Yeast and Minimal Inhibitory Concentration of Amphotericin B and Voriconazole
The American Type Culture Collection (ATCC) strain of C. albicans (ATCC 90028) was utilized. The YeastOne method determined MICs of voriconazole and amphotericin B. 1 × MICs for voriconazole and amphotericin B for C. albicans were 0.008 μg/ml (the Clinical and Laboratory Standards Institute (CLSI) quality control range of 0.004–0.016 μg/ml) and 0.25 μg/ml (the CLSI range of 0.125–0.5 μg/ml), respectively. Given the known toxicity of amphotericin B, the lower limit of its MIC range (0.25 μg/ml) was used as the baseline for determining the multiples of MIC, while the upper limit (5 μg/ml) was chosen for the highest MIC tested in the study. To facilitate a fair comparison between voriconazole and amphotericin B, similar MIC multiples were used for voriconazole.
Corneoscleral Rim and Simulation of Contamination During Retrieval
Culture-negative corneoscleral rims were divided into standardized segments, with each weighing approximately 0.03 g. Each piece was immersed in a 5-ml suspension containing 104 colony-forming units per milliliter (CFU/ml) of C. albicans for 2 h at room temperature. The contaminated tissue segments were then rinsed in sterile phosphate-buffered saline (PBS) to remove fungi superficially adhering to the tissue surface. Following the rinse, the contaminated tissues were incubated in Optisol-GS, either with or without an antifungal supplement, for further examination.
Measurement of Antifungal Efficacy in Optisol-GS Samples
For each Optisol-GS sampling, a 100-μL sample was diluted with sterile water at a 1:10 ratio to minimize potential antifungal carryover effects, and then cultured on Sabouraud agar. All culture plates were incubated at 35 °C for 36 h, after which viable colony counts were recorded. The detection limit was defined as the lowest number of colonies detectable by the assay, set at 10 CFU/ml. At least two separate experiments, each with triplicate cultures, were conducted for all conditions and time points.
Sterility Measurement of Corneoscleral Tissues by Direct Tissue Culture and Histopathology
After a 14-day hypothermic incubation, corneoscleral tissues stored in Optisol-GS with varying antifungal supplementation timing were removed from the vials and rinsed with sterile PBS to eliminate fungi superficially adhering to surfaces. The corneoscleral tissues were evaluated in two ways: (1) placed in 5 ml of Tryptic Soy Broth (TSB) and incubated at 37 °C for 48 h to assess fungal growth based on media clarity; and (2) fixed in 4% formalin for 24 h, embedded in paraffin and subjected to periodic acid–Schiff (PAS) staining on 5-µm-thick sections to detect fungi. All evaluations were performed in triplicate, and the results were averaged.
Data Analysis
The logarithmic scale of the mean microbial concentration (log10 CFU/ml) and its standard deviation (SD) were computed. log10 reduction was calculated using the formula: log10 CC(T0) − log10 CC(Tt), where CC(T0) represents the colony counts per milliliter at the initial time (time of inoculation), and CC(Tt) represents the colony counts per milliliter at a later time t. Positive log10 reduction values indicate a decrease in colony count relative to the baseline count (indicating microbial killing), while negative values suggest an increase in colony count (indicating microbial growth). The time-kill plot graphically represents the mean log10 colony counts per milliliter over time for various timings of antifungal supplementation.
Mann–Whitney U test compared the log10 reductions between different temperature exposures at the same MIC after a 7-day incubation period. A P value < 0.05 indicated statistical significance.
Results
Killing Efficacy of Antifungal Agents in Optisol-GS Without or with Room Temperature Exposure (Regimen A vs. Regimen B) after 7 days of Incubation
The killing efficacy of antifungal agents, as reflected by the log10 reduction values, is presented in Table 1. In the control samples without antifungal agents, the log10 reduction values were consistently negative by day 7, indicating ongoing growth of C. albicans under both regimens A and B. However, in samples supplemented with antifungal agents, the log10 reduction values increased proportionally with higher multiples of MIC. This suggests that the addition of antifungal agents reduces the growth of C. albicans in a concentration-dependent manner. There were no significant differences in log10 reduction values between regimen A and B for Optisol-GS supplemented with either voriconazole or amphotericin B at similar MIC multiples after a 7-day incubation period (P > 0.05, Mann–Whitney U test). Over the 7-day incubation under both regimens A and B, positive log10 reduction values for voriconazole were observed only at 20 × MIC, whereas for amphotericin B, positive values were noted at 10 × and 20 × MICs. However, even at 20 × MIC of voriconazole or amphotericin B, less than a 95% reduction in C. albicans growth in the Optisol-GS samples was observed (log10 reduction of 0.72–1.04 and 0.88–1.26 for voriconazole and amphotericin B, respectively).
Table 1
log10 reduction of C. albicans in Optisol-GS supplemented with voriconazole or amphotericin B on day 7
Log10 reduction of C. albicans after 7-day incubation [Mean ± Standard deviation]
Regimen A (2–8 °C)
Regimen B (room temperature exposure)
P value
Control
− 1.13 ± 0.06
− 1.14 ± 0.03
> 0.99
Voriconazole
2 × MIC
− 0.44 ± 0.05
− 0.46 ± 0.05
0.79
5 × MIC
− 0.20 ± 0.01
− 0.37 ± 0.05
0.1
10 × MIC
− 0.15 ± 0.08
− 0.26 ± 0.06
0.4
20 × MIC
0.88 ± 0.1
0.88 ± 0.16
0.2
Amphotericin B
2 × MIC
− 0.43 ± 0.01
− 0.45 ± 0.06
> 0.99
5 × MIC
− 0.02 ± 0.06
− 0.01 ± 0.08
> 0.99
10 × MIC
0.31 ± 0.10
0.14 ± 0.09
0.2
20 × MIC
1.11 ± 0.15
1.0 ± 0.12
> 0.99
log10 reduction values are positive when colony counts decrease compared to baseline counts (indicating microbial killing) and negative when colony counts increase (indicating microbial growth). A Mann–Whitney U test was used to evaluate the significance of differences in log10 reduction between regimen A and regimen B at the same MIC. The resulting P values indicate the level of statistical significance
Killing Efficacy of Antifungal Agents in Optisol-GS with Different Timings of Supplementation After 14 days of Incubation
The initial concentration of C. albicans on day 0 in Optisol-GS with contaminated corneoscleral rim tissue was 3.27 ± 0.05 log10 CFU/ml (Fig. 2). The effectiveness of 20 × MIC of voriconazole or amphotericin B in Optisol-GS was assessed daily over a 14-day incubation period in the following four groups. This 14-day observation period was chosen because it aligns with the recommended shelf life of corneal storage in Optisol-GS. Viable colonies of C. albicans were detected at all time points in the vials supplemented with a single addition of either voriconazole or amphotericin B (groups A and D in Fig. 2). In contrast, when an additional dose of antifungal agents was introduced on either day 2 or day 6, the viable colony count of C. albicans in Optisol-GS samples decreased to below the detection limit by day 14 (log10 reduction of 2.26 ± 0.05 for voriconazole and 2.25 ± 0.04 for amphotericin B, respectively).
Fig. 2
Growth of C. albicans in Optisol-GS under different timing of antifungal additions at 20 × MIC. a Time-kill plot over time in vials supplemented with amphotericin B at 20 × MIC. b Time-kill plot over time in vials supplemented with voriconazole at 20 × MIC. The 4 groups of antifungal additions are represented as follows: day 0 (group A), days 0 and 2 (group B), days 0 and 6 (group C), or day 6 only (group D). The dotted line represented the limit of detection limit at 10 colony-forming units/ml. It is noteworthy that there is an overlap between most values of the control and group D, as well as between group A and group C, throughout the period from day 0 to day 14
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Sterility Measurement of Corneoscleral Rim After 14 days of Incubation in Optisol-GS with Antifungal Supplementation
Table 2 displays the recovery rate of C. albicans from contaminated corneoscleral tissues after a 14-day incubation at 2–8 °C in Optisol-GS supplemented with 20 × MIC of voriconazole or amphotericin B. Compared to groups with single antifungal supplementation (group A and D), only in group B and C, where an additional dose of antifungal agents was administered on either day 2 or 6 (groups B and C), all corneoscleral tissues tested negative for the fungus. The histopathological examination of the contaminated corneoscleral tissues after a 14-day incubation in antifungal-supplemented Optisol-GS under hypothermic conditions corroborated the direct tissue culture results. Despite a single dose of antifungal supplementation in Optisol-GS on day 0 or 6, positive PAS staining, indicating the presence of fungi, was still observed in the corneoscleral tissues (Fig. 3). However, in groups where an extra dose of antifungal agents was added on either day 2 or 6, no fungi were detected by PAS staining.
Table 2
Recovery of C. albicans from direct tissue culture of contaminated corneoscleral rims in antifungal supplemented Optisol-GS after 14-day incubation at 2–8 °C
Timing of antifungal supplementation
Recovery rate
20 × MIC
Voriconzole
Group A
(day 0)
75%
Group B
(day 0 and day 2)
0
Group C
(day 0 and day 6)
0
Group D
(day 6)
100%
20 × MIC
Amphotericin B
Group A
(day 0)
75%
Group B
(day 0 and day 2)
0
Group C
(day 0 and day 6)
0
Group D
(day 6)
100%
All determinations were performed in triplicate, and the results were averaged
MIC minimal inhibitory concentration
Fig. 3
Representative histological images with periodic acid–Schiff (PAS) staining of corneoscleral tissues following a 14-day incubation. a, b Negative PAS staining on contaminated corneoscleral tissues incubated with 20 × MIC voriconazole, with double supplementation on day 0 and day 6. c, d Positive PAS staining (indicated by arrows) highlighting the presence of Candida organisms on contaminated corneoscleral tissues incubated with 20 × MIC voriconazole, with a single supplementation on day 0. The scale bar represents 20 μm
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Discussion
Post-EK fungal infections are challenging because of their severity and deep-seated nature [1, 16]. Multiple species have been reported in post-EK fungal infections, with Candida species comprising the majority of culture-proven cases (Table S1). Efforts to prevent tissue contamination have led to improvement in disinfection protocols and reduced warming time during tissue processing [4]. Additionally, in vitro studies have demonstrated the effectiveness of antifungal supplementation in hypothermic storage [5‐9, 17, 18], underscoring its potential in preventing fungal infections in EK procedures. Furthermore, recent studies suggest that antifungal supplementation could be cost-effective for EK surgeries [14, 15]. However, there is variability in the reported effective concentrations of antifungal agents across studies, likely due to variations in experimental designs. Literature review reveals a lack of consensus on dosing and temperature influence of antifungals against Candida species, the most commonly encountered fungal pathogen in post-EK infections (Table 3).
Table 3
Review of studies regarding the efficacy of antifungal agents against Candida species in hypothermic storage
To accurately replicate real-world scenarios before systemic supplementation of antifungal agents, appropriate models are imperative. Unlike most preceding studies [5‐7], our research utilized a novel simulation model that directly immerses corneoscleral tissue in fungal inoculum, mimicking tissue contamination during retrieval. We evaluated the antifungal efficacy using this model in conjunction with the warming protocol during eye bank processing. Our findings indicated a concentration-dependent effect of antifungal agents, consistent with other studies [5, 6, 9]. However, even at 20 × MIC (0.16 μg/ml of voriconazole or 5 μg/ml of amphotericin B), killing efficacy between day 0 and day 7 was less than 95%. Reviewing existing literature, we identified only two studies employing direct tissue inoculation. Tran et al. found that amphotericin B at a concentration of 0.255 μg/ml did not completely eradicate Candida species from corneoscleral rims over 48 h [19]. Conversely, Giurgola et al. observed a notable reduction in Candida colonies after 5 or 10 days of hypothermic incubation in Kerasave, which contained amphotericin B at 2.5 μg/ml [17]. Comparing our results with studies using inoculated storage medium [5, 6, 9], amphotericin B at similar concentrations was less effective, likely due to differences in inoculation models and inoculum concentrations.
Tissue warming during examination and processing may influence fungal growth and antifungal agent depletion [4, 7]. However, our findings indicated no significant difference between hypothermic and room temperature exposure groups, suggesting controlled warming times may not substantially impact antifungal efficacy. This contrasts with the findings of a study by Layer et al. [6], where amphotericin B was more effective than voriconazole in reducing Candida species. Unlike their direct inoculation of the storage medium, our research utilized tissue. Given that tissue penetration is a crucial factor in assessing antifungal efficacy, the dynamics and absolute concentration of an antifungal drug within tissue or storage medium may vary considerably [20]. Consequently, it is plausible that differences in methodologies, especially the direct inoculation of storage medium versus the use of tissue, contribute to variations in observed results.
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To address the suboptimal reduction in yeast growth observed with a single antifungal agent addition in our first experiment, we designed a second experiment assessing the impact of supplementation timing on antifungal efficacy during the commonly practiced 2-week hypothermic incubation. We selected day 2 as one of the time points on the basis of previous studies indicating that the time-kill plot tends to deviate from its initial declining trend around this day [6, 7]. Day 6 was chosen to simulate the last opportunity for supplementation prior to tissue release, which typically occurs within 7 days of procurement. Moreover, we evaluated tissue sterility to confirm the yeast elimination from contaminated tissue. Our study showed that additional supplementation of either voriconazole or amphotericin B led to over 99% yeast growth reduction. The ineffectiveness of a single supplementation may stem from gradual drug degradation or consumption following microbial killing. Previous studies in a 28-day organ culture storage found voriconazole maintained a stable concentration after initially declining to 80% by day 7. In contrast, amphotericin B retained only 60% of its original concentration by the end of the storage period [21]. Experience from other fields suggests amphotericin B degrades within 2 days under warm storage conditions [22], yet such data for hypothermic storage are currently lacking. These findings imply that antifungal agent degradation and consumption over time may limit efficacy with a single supplementation. The additional supplementation of antifungal agents at appropriate intervals appears necessary to sustain yeast growth reduction during hypothermic storage. Further research is needed to evaluate antifungal degradation and stability, specifically under hypothermic storage conditions.
The current study has several limitations. First, we tested only one strain of C. albicans, which may limit the generalizability of our findings. This focused approach aimed to reduce variability and concentrate on the efficacy of antifungal agents under different temperature conditions and drug supplementation timings. Second, we did not conduct an extensive safety study. Although the highest concentration of amphotericin B used in our study (5 μg/ml) was below the toxic level reported in previous research [6], a comprehensive safety evaluation is crucial prior to the clinical implementation. Lastly, we did not directly monitor the stability of the antifungal additives during the storage period. Although beyond our study’s scope, investigating degradation and stability of antifungals over time is important to confirm their effectiveness throughout the storage duration. Addressing these limitations in the future would enhance our understanding of antifungal efficacy, safety, and stability under hypothermic storage conditions, offering insights to improve tissue preservation protocols and preventing post-EK fungal infections.
Conclusions
Post-EK fungal infections pose significant challenges in clinical practice, underscoring the need for effective preventive measures. Our approach stands out for its close alignment with actual eye banking practices, providing insights more directly applicable to clinical settings. Our results demonstrate that adhering to a regulated warming protocol does not undermine antifungal efficacy. This finding holds particular significance, given the widespread use of warming procedures during tissue inspection and processing in eye banks. Additionally, our study underscores the limitations of relying solely on single antifungal additions to eliminate fungal loads during routine hypothermic storage. This understanding is pivotal for informing clinical implementation and guiding future investigations into drug–tissue penetration and interaction.
Acknowledgements
We would like to extend our heartfelt gratitude to the donors and their families for their generous contributions.
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Authorship
All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.
Declarations
Conflict of Interest
Hsin-Yu Liu, Pao-Yu Chen, Hsiao-Sang Chu, Ya-Ting Chiu, Yee-Chun Chen, and Fung-Rong Hu confirm that they have no competing interests to declare.
Ethical Approval
The study received approval from the Institutional Research Ethics Board of National Taiwan University Hospital (IRB No. 201904036RIND). All procedures were performed in accordance with the tenets of the Declaration of Helsinki of 1964 and its later amendments.
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc/4.0/.
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