To determine the concentration of amphotericin B that would be both effective against Candida albicans contamination and safe for corneal endothelial cells (CECs) in cold storage conditions.
Triplicate media cultures were inoculated with 105 colony-forming units (CFUs)/mL of C. albicans (American Type Culture Collection 10231), supplemented with amphotericin B (0–20 μg/mL), stored in cold conditions (2°C–8°C) for 72 hours, and analyzed quantitatively for CFUs. C. albicans concentration in each sample was determined initially and after 6, 24, 48, and 72 hours of storage. CEC mitochondrial function (oxygen consumption rate), apoptosis, and necrosis were examined in donor corneas after 7 days of amphotericin B exposure and compared with untreated controls. CEC viability was also examined by calcein-AM staining and Fiji segmentation after 72 hours or 2 weeks of amphotericin B exposure to mimic potential eye bank practices.
Amphotericin B concentrations of 1.25, 2.5, and 5.0 μg/mL resulted in 0.47, 1.11, and 1.21 log10 CFU reduction after only 6 hours of cold storage and continued to decrease to 3.50, 3.86, and 4.49 log10 reductions after 72 hours, respectively. By contrast, amphotericin B 0.255 µg/mL showed only 1.01 log10 CFU reduction after 72 hours of incubation. CEC mitochondrial function and viability did not differ in donor corneas exposed to amphotericin B ≤2.59 μg/mL compared with the controls.
Optimal efficacy of amphotericin B against C. albicans is achieved in cold storage conditions at concentrations ≥1.25 μg/mL, and 2.5 μg/mL reduces Candida contamination by >90% after 6 hours of cold storage without sacrificing CEC health.
*Lions VisionGift, Portland, OR;
†Department of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, Department of Ophthalmology and Visual Sciences, Iowa City, IA;
‡Iowa Lions Eye Bank, Coralville, IA;
§Alchilife Srl, Ponte San Nicolò, Padova, Italy;
¶Cornea Services, Devers Eye Institute, Portland, OR; and
‖Cornea Research Center, Department of Ophthalmology and Visual Sciences, Iowa City, IA.
Correspondence: Mark A. Greiner, MD, Department of Ophthalmology and Visual Sciences, Cornea and External Diseases, University of Iowa Carver College of Medicine, 200 Hawkins Drive, Iowa City, IA 52242 (e-mail: email@example.com).
Supported in part by a research grant from the Eye Bank Association of America. Donated corneas used in this study were provided by Iowa Lions Eye Bank and Lions VisionGift.
J. D. Tóthová, L. Giurgola, and C. Gatto are employees of AL.CHI.MI.A.S.R.L and were contracted to perform the efficacy studies of amphotericin B. The authors have no conflicts of interest to disclose.
K. D. Tran, B. T. Aldrich, and J. D. Tóthová contributed equally to this study.
Received March 13, 2019
Received in revised form April 18, 2019
Accepted April 21, 2019