The number of mean viable fungal colonies on days 2, 7, and 14 in vials of Optisol-GS supplemented with 0.06-, 0.12-, and 0.255-μg/mL concentrations of amphotericin B were compared between light-exposed and light-protected conditions in the efficacy study. CFU indicates colony-forming units.
Customize your JAMA Network experience by selecting one or more topics from the list below.
Duncan K, Parker J, Hoover C, Jeng BH. The Effect of Light Exposure on the Efficacy and Safety of Amphotericin B in Corneal Storage Media. JAMA Ophthalmol. 2016;134(4):432–436. doi:10.1001/jamaophthalmol.2016.0008
The proportion of postkeratoplasty fungal infections is rising steadily. However, the most commonly used corneal storage medium in the United States, Optisol-GS, does not contain an antifungal additive.
To determine the lowest concentration of amphotericin B supplementation in Optisol-GS that will eliminate fungal contaminants effectively without resulting in toxic effects to the cornea and to determine what role light exposure plays in the efficacy and safety of amphotericin B supplementation.
Design, Setting, and Materials
An in vitro laboratory efficacy study measured fungal colony growth in 10 vials of Optisol-GS supplemented with different concentrations of amphotericin B after inoculation with Candida albicans in light-exposed and light-protected conditions. Two vials each were supplemented with amphotericin B at concentrations of 0.06, 0.12, or 0.225 μg/mL; the remaining 2 vials received no C albicans inoculation and no antifungal supplementation (negative controls). After 24 hours, 1 vial from each pair was exposed to light for the remainder of the study. On days 2, 7, and 14, 1 mL of solution was removed from each vial and incubated at 36°C for 48 hours. In a separate safety study, 12 pairs of corneas were divided between amphotericin B supplementation and the control condition; 4 corneas each received the different amphotericin B concentrations. An additional 4 pairs of corneas were stored in the 0.225-μg/mL concentration, and 1 cornea from each pair was exposed to light for the duration of the study. Data were collected November 16, 2014, and analyzed from November 16 to 18, 2014, for the efficacy study; they were collected from April 14 to May 27, 2015, and analyzed from May 28 to 30, 2015, and on December 23, 2015, for the safety study.
Main Outcomes and Measures
Fungal colony growth was measured from the Optisol-GS vials. Corneal endothelial cell density, endothelial cell viability, and epithelial toxic effects were measured in stored corneas.
In the efficacy study, Optisol-GS supplemented with concentrations of 0.06 and 0.12 μg/mL of amphotericin B eliminated all fungal contaminants by day 7 and reduced fungal growth on day 2 by a mean of 3.5 colony-forming units (95% CI, −6.19 to 13.20 colony-forming units; P = .34), a 77.8% decline compared with the postoperative controls. Optisol-GS supplemented with the 0.255-μg/mL concentration of amphotericin B eliminated all fungal contaminants by day 2. In the safety study, no evidence was found of toxic effects to the cornea in corneas stored in Optisol-GS supplemented with amphotericin B at any concentration compared with paired controls. No difference in the efficacy or safety of the light-exposed compared with light-protected amphotericin B–supplemented Optisol-GS was identified.
Conclusions and Relevance
In this study, Optisol-GS supplemented with a 0.255-μg/mL concentration of amphotericin B effectively eliminated fungal contaminants within 48 hours and did not result in added toxic effects to the cornea. These results do not prove that amphotericin B should be added to Optisol-GS; larger-scale studies and cost-benefit analyses need to be completed. Given the increasing incidence of postkeratoplasty fungal infection, however, the addition of amphotericin B to Optisol-GS deserves further investigation.
Fungal infections after corneal transplants have been reported in only 0.014% of transplants from 2007 to 2010.1 However, fungal infection is often visually devastating when it occurs. At present, the most commonly used corneal storage medium in the United States, Optisol-GS (Bausch & Lomb, Inc), contains antibiotics, but no antifungal. The most commonly used corneal storage medium in Europe, in contrast, contains antibiotics and the antifungal amphotericin B.2
The introduction of Optisol-GS, which contains gentamicin and streptomycin sulfate, in the early 1990s resulted in a dramatic decline in rates of postkeratoplasty bacterial infection.1 Since that time, the proportion of fungal infections after corneal transplant has been steadily climbing from 10% in 1991 to 63% from 2007 to 2010.1,3Candida albicans is the most common fungal species responsible for these infections.1,4-6 With the increasing popularity of endothelial keratoplasty, fungal infections have been observed almost twice as often after endothelial keratoplasty compared with penetrating keratoplasty.1
These observed trends led the Eye Bank Association of America (EBAA) to form a subcommittee in 2010 to investigate the merit and feasibility of adding antifungal supplements to corneal storage media in the United States.1 This subcommittee reviewed the available data and confirmed “an increasing trend in the incidence of postkeratoplasty fungal infection…”1(p153) but did “not recommend that storage media should be supplemented with an antifungal at this time.”1(p154) They cited relevant factors leading to that decision to be the “lack of sufficient evidence regarding the efficacy, safety and cost of antifungal supplementation.”1(p154)
To provide additional evidence of the safety and efficacy of antifungal supplementation, a previous study7 evaluated amphotericin B and voriconazole and found that amphotericin B was very effective at eliminating Candida species from Optisol-GS, whereas voriconazole was not. The primary aim of the present study was to determine the lowest concentration at which amphotericin B may eliminate fungal contaminants effectively without introducing any toxic effects to the cornea. We also sought to determine what role light inactivation may play in the efficacy and safety of amphotericin B as an antifungal supplement in Optisol-GS. Amphotericin B is degraded by light and traditionally has been stored in light-protected conditions. Data suggest that the antifungal activity of amphotericin B declines within 24 to 96 hours of exposure to light.8-10 We sought to determine whether exposing Optisol-GS supplemented with amphotericin B to light would allow it enough time to exert its antifungal activity but would degrade the drug early enough to reduce potential toxic effects to the cornea.
Question: What is the lowest concentration of amphotericin B that effectively and safely eliminates fungal contaminants in corneal storage media?
Findings: Corneal storage media inoculated with Candida albicans and supplemented with amphotericin B at a concentration of 0.255 μg/mL effectively eliminated all fungal growth by 48 hours. The differences in endothelial cell count, percentage of intact epithelium, and percentage of nonviable endothelial cells in corneas stored in amphotericin B–supplemented vs unsupplemented storage media were not significant.
Meaning: As the incidence of fungal infection after corneal transplant rises, the addition of an amphotericin B to corneal storage media should be considered.
Data were collected from November 12 to 16, 2014, for the efficacy study and from April 14 to May 27, 2015, for the safety study. Ten vials of Optisol-GS were divided into 5 groups. The first group was supplemented with amphotericin B at a concentration of 0.06 μg/mL; the second, 0.12 μg/mL; and the third, 0.255 μg/mL. Amphotericin B ophthalmic solution, 0.15%, was obtained from Leiter’s Compounding Pharmacy and serially diluted in Optisol-GS to create the appropriate concentrations for each group.
The fourth group was not supplemented with amphotericin B and served as our positive control group. Groups 1 to 4 were all inoculated with Candida albicans. The inoculum was prepared by incubating a strain of C albicans (American Type Culture Collection 10231) at 36°C for 24 hours on a Sabouraud agar plate. Colonies were removed from the plate and suspended in sterile saline to obtain turbidity consistent with the 0.5 McFarland standard (1 × 106 to 5 × 106 colony-forming units [CFUs]/mL).11 The fungal concentration was confirmed by serial dilution, subculture, and counts of viable fungal CFUs. An appropriate volume of this suspension was added to each Optisol-GS vial to create a suspension of 2.5 × 106 CFUs/mL. The fifth group of Optisol-GS vials was not supplemented with amphotericin B and not inoculated with C albicans (negative control group). The institutional review board of the University of Maryland waived approval for this laboratory study.
All vials were refrigerated at 4°C and were protected from light for the first 24 hours of the study. At 24 hours, 1 vial from each group was uncovered and exposed to light for the remaining duration of the study. On days 2, 7, and 14, 1 mL of solution was removed from each vial. A 10-μL sample was taken from each 1 mL and plated directly on a Sabouraud agar plate. An additional 100-μL sample was also taken from each 1 mL and diluted with Optisol-GS 1:10 (to minimize antifungal carryover) and then placed on a separate Sabouraud agar plate. The plates were incubated at 36°C for 48 hours, after which time fungal CFU counts were performed. A sample from a colony on each plate (if available) was examined with a light microscope to ensure the microorganisms growing were consistent with C albicans, and not any bacterial contaminants.
Twelve pairs of corneas that were not suitable for transplant owing to the donor’s medical history were obtained from SightLife, Inc, and divided into 3 groups of 4 pairs each. One cornea from each pair was stored in Optisol-GS supplemented with 0.06-, 0.12-, and 0.225-μg/mL concentrations of amphotericin B in the first, second, and third groups, respectively. The second cornea of each pair served as a control and was stored in unsupplemented Optisol-GS. All corneas were stored at 4°C consistent with standard protocol of the Eye Band Association of America (EBAA). The corneas were evaluated on days 0, 7, and 14 for endothelial cell density via specular microscopy and for the percentage of intact epithelium via slitlamp examination. These evaluations were conducted according to standard EBAA protocol by SightLife, Inc, technicians who were masked to the identity of each cornea. Paired t tests were used to compare the mean change in endothelial cell density and the percentage of intact epithelium from days 0 to 14 for the amphotericin B–supplemented and control corneas.
On day 16, the corneas were stained with 0.4% trypan blue and 1% alizarin red according to the staining protocol published by Park et al.12 Trypan blue stains severely damaged or dead endothelial cells, whereas alizarin red stains cell borders and areas of denuded Descemet membrane. After staining, the corneas were trephinated and placed on a microscope slide with the endothelium facing up. A slide cover was placed over each cornea and they were examined with light microscopy at a magnification factor of ×40. Five representative photographs were taken of different fields within the central area of each cornea. The numbers of viable and nonviable endothelial cells were counted in each photograph, which allowed for calculation of the mean percentage of nonviable endothelial cells for each cornea.
An additional 4 pairs of corneas were used to evaluate the effect of light exposure on the corneal toxicity of amphotericin B. All corneas in each of these pairs were stored in Optisol-GS supplemented with a 0.255-μg/mL concentration of amphotericin B. All of the corneas were protected from light for the first 24 hours of the study. After 24 hours, 1 cornea from each pair was exposed to light while the other cornea was protected from light for the duration of the study. Evaluation of endothelial cell density, percentage of intact epithelium, and vital dye staining were performed as described above.
Data were analyzed from November 16, 2014, to November 18, 2015, for the efficacy study, and May 28 to 30, 2014, and on December 23, 2015, for the safety study. Mean fungal CFU growth from the amphotericin B–supplemented Optisol-GS vials was compared with mean CFU growth from the unsupplemented control vials using independent t tests. We used paired t tests to compare the percentage of nonviable endothelial cells between the amphotericin B–supplemented and control corneas and to calculate differences in mean change in endothelial cell density, percentages of intact epithelium from days 0 to 14, and the percentage of nonviable endothelial cells between the light-exposed and the light-protected corneas.
We found no fungal colony growth on day 7 or 14 from the Optisol-GS vials supplemented with amphotericin B at any concentration. Fungal colony growth occurred on day 2 at the lower concentrations of amphotericin B supplementation; however, growth was reduced by a mean of 3.5 CFUs (95% CI, −6.19 to 13.20 CFUs; P = .34), which corresponds to a 77.8% reduction when compared with positive controls. No fungal colony growth was found on any day in Optisol-GS vials supplemented with a 0.255-μg/mL concentration of amphotericin B (Figure). No difference in fungal growth between the light-exposed and light-protected corneas at any concentration of amphotericin B supplementation on any day of the study was identified.
In the first set of 12 pairs of corneas, we found no difference in the mean change in endothelial cell density between the amphotericin-supplemented and control corneas at our lowest (0.06-μg/mL) and highest (0.255-μg/mL) concentrations of amphotericin B. The decrease in endothelial cell density was greater in the control group compared with the corneas stored in Optisol-GS supplemented with the 0.12-μg/mL concentration of amphotericin B (Table 1). We found no difference in the change in the percentage of intact epithelium between the control and amphotericin B–supplemented corneas at any concentration (Table 1). We also found no difference in the percentage of viable endothelial cells on day 16 between the control and amphotericin B–supplemented corneas at any concentration (Table 1). One of the 4 pairs of corneas in the second group (supplemented with the 0.12-μg/mL concentration of amphotericin B) were not included in data analysis owing to an error in the staining protocol for this pair. In the second set of corneas, which were all stored in Optisol-GS supplemented with the 0.255-μg/mL concentration of amphotericin B, no difference in the mean change in endothelial cell density, percentage of nonviable endothelial cells, or percentage of intact epithelium was identified between the light-exposed and the light-protected corneas (Table 2).
The rate of fungal infection after corneal transplant has increased significantly in recent years. The most recent data generated from the online adverse reaction reporting system presented to the Medical Advisory Board of the EBAA in June 2015 confirms that these rates continue to rise (EBAA Medical Advisory Board meeting, written communication, June 6, 2015). From 2010 to 2014, the incidence of postkeratoplasty fungal endophthalmitis has increased 2.5-fold, and the incidence of postkeratoplasty fungal keratitis has increased 4-fold. All cases of postkeratoplasty fungal infection reported in 2014 had cultures that grew Candida species with the exception of 1 case (EBAA Medical Advisory Board meeting, written communication, June 6, 2015). Keyhani et al5 found that the incidence of postkeratoplasty fungal infection in recipients of donor corneas that had positive corneoscleral rim cultures was as high as 14%, demonstrating the importance of obtaining corneoscleral rim cultures.
Another clear change represented in the data is the increased incidence of infection after endothelial keratoplasty compared with penetrating keratoplasty. Endothelial keratoplasty allows for a unique surgical situation whereby fungal elements that normally could be cleared from the anterior chamber are sequestered and proliferate in the lamellar interface between the donor lenticule and host cornea. Of all cases of postkeratoplasty endophthalmitis reported to the EBAA in 2014, 73% occurred in cases of endothelial keratoplasty (EBAA Medical Advisory Board meeting, written communication, June 6, 2015). Eighty-eight percent of fungal keratitis reported after corneal transplant in 2014 also occurred in cases of endothelial keratoplasty (EBAA Medical Advisory Board meeting, written communication, June 6, 2015). Numerous case reports in the literature describe aggressive fungal infections after endothelial keratoplasty that are often refractory to conservative management and require therapeutic penetrating keratoplasty13-18
This study confirmed the efficacy and safety of amphotericin B as an antifungal supplement in Optisol-GS. Although all concentrations of amphotericin B effectively eliminated fungal contaminants within 7 days, only the 0.255-μg/mL concentration eliminated all fungal contaminants within 2 days. None of the concentrations of amphotericin B showed evidence of toxic effects to the cornea. Because many corneal surgeons want to use tissue that has been stored for the shortest time possible, an antifungal supplement that acts quickly is preferred. As such, we believe that 0.255 μg/mL would be the ideal concentration for amphotericin B supplementation, given its rapid efficacy and safety.
This concentration is very similar to the concentration of amphotericin B used in corneal storage media in Europe (0.25 μg/mL).6 That the ideal concentration of antifungal supplementation should be the same on both continents may seem obvious, but one must remember that the corneal storage processes in the United States and Europe differ greatly, with European eye banks using predominately warm storage whereas eye banks in the United States use hypothermic storage.2 Microbial growth and pharmacokinetics are highly influenced by temperature; therefore the concentrations of ideal antifungal supplementation may differ greatly between both processes.
We did not find any evidence that light exposure influences the efficacy or safety of amphotericin B as an antifungal supplement in Optisol-GS. We did not attempt to measure the intensity or wavelength of the light to which the Optisol-GS vials were exposed, which may represent a limitation of the study. The vials were exposed to standard ambient lighting in the microbiology laboratory and eye bank. Should amphotericin B be added to Optisol-GS in the future, no extra efforts likely would be required to keep the storage medium protected from light; however, additional testing with specified light intensity and wavelength may be warranted to confirm this finding.
Given its increasing incidence and devastating visual outcomes, postkeratoplasty fungal infection is a growing problem. The EBAA subcommittee on fungal infection after corneal transplant was previously unable to recommend the addition of an antifungal to corneal storage media in the United States owing to a lack of evidence regarding the safety and efficacy of these supplements.1 Our data alone do not prove that amphotericin B should be added to Optisol-GS, because larger studies and cost-benefit analysis are needed. However, we hope that our study has provided enough evidence to warrant reconsideration of this issue. We believe that current developments in postkeratoplasty fungal infection along with our data and the low cost of amphotericin B merit reevaluation of the addition of antifungal supplements to Optisol-GS.
Corresponding Author: Bennie H. Jeng, MD, MS, Department of Ophthalmology and Visual Sciences, University of Maryland School of Medicine, 419 W Redwood St, Ste 470, Baltimore, MD 21201 (firstname.lastname@example.org).
Submitted for Publication: October 1, 2015; final revision received December 24, 2015; accepted January 1, 2016.
Published Online: February 25, 2016. doi:10.1001/jamaophthalmol.2016.0008.
Author Contributions: Drs Duncan and Jeng had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Duncan, Parker, Jeng.
Acquisition, analysis, or interpretation of data: Duncan, Hoover, Jeng.
Drafting of the manuscript: Duncan, Parker.
Critical revision of the manuscript for important intellectual content: Hoover, Jeng.
Statistical analysis: Duncan, Jeng.
Obtained funding: Duncan, Jeng.
Administrative, technical, or material support: All authors.
Study supervision: Hoover, Jeng.
Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.
Funding/Support: This study was supported by a Richard L. Lindstrom/Eye Bank Association of America research grant. SightLife, Inc, provided the research corneas.
Role of the Funder/Sponsor: The funding source had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Previous Presentations: This paper was presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology; May 4, 2015; Denver, Colorado; the Annual Meeting of the Eye Bank Association of America; June 6, 2015; Atlanta, Georgia; and the Annual Meeting of the Ocular Microbiology Immunology Group; November 13, 2015; Las Vegas, Nevada.
Additional Contributions: Technicians at SightLife, Inc, conducted evaluations of endothelial cell density and percentage of intact epithelium. Osamah Saeedi, MD, and Braxton D. Mitchell, PhD, University of Maryland School of Medicine, assisted with statistical analysis of the data and the accurate calculation of CIs for the efficacy portion of the data because they have expertise in this area. They were not compensated for this work.
Create a personal account or sign in to: