Efficacy and Safety of Various Amphotericin B Concentrations on Candida albicans in Cold Storage Conditions

Abstract

Purpose: 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. Methods: 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. Results: 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. Conclusions: 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.

Cost-Effectiveness of Antifungal Supplementation of Corneal Cold Storage Media

“…24,26,34 At 2.5 mg/ml, there was a 99.8% reduction with no significant effect on corneal endothelial cell health. 26 At 4 mg/ml, there was a trend for decreased endothelial cells but minimal changes in mitochondrial function, and at significantly higher concentrations of 40 mg/ml, there was a significant reduction in endothelial cell density. 22,26 Potential toxicity could be mitigated by using low concentrations of antifungals in the corneal storage media and eluting the grafts.…”

“…2,8,23,[30][31][32][33] In particular, amphotericin B 0.255 mg/ml dose was associated with an 85% reduction in fungal colonies after 48 hours, with no evidence of corneal toxicity. 22,26 At 1.25 mg/ml, there was a 98.3% reduction, but there may be a transient and reversible functional impairment of endothelial cell function. 24,26,34 At 2.5 mg/ml, there was a 99.8% reduction with no significant effect on corneal endothelial cell health.…”

“…22,26 At 1.25 mg/ml, there was a 98.3% reduction, but there may be a transient and reversible functional impairment of endothelial cell function. 24,26,34 At 2.5 mg/ml, there was a 99.8% reduction with no significant effect on corneal endothelial cell health. 26 At 4 mg/ml, there was a trend for decreased endothelial cells but minimal changes in mitochondrial function, and at significantly higher concentrations of 40 mg/ml, there was a significant reduction in endothelial cell density.…”

“…In vitro studies have demonstrated a 96% to 100% effectiveness for amphotericin B against Candida, 70% for voriconazole, and 96.5% for caspofungin. 2,8,[22][23][24][25][26] The probability sensitivity analyses (PSAs) were conducted by varying the current fungal infection rates from 86% to 99.9% for amphotericin B, 65% to 99.9% for voriconazole, and 77% to 99.9% for caspofungin based on in vitro studies on the rates of antifungal resistance (Table 2). 2,8,[22][23][24][25][26] To account for potential endothelial toxicity with the addition of antifungals, the risk of graft failure with antifungals was increased by a theoretical 5% from the current rates to evaluate the CEA of antifungal supplementation under the most stringent conditions (Table 1).…”

Survival of Candida parapsilosis Isolates in Cold Storage Conditions: Recovery in Cultures After 10-Years of Storage

“…Transplante edilecek dokuların soğuk saklama koşullarında kontaminasyona yol açabilen Candida türlerinin kornea nakillerinde postkeratoplasti enfeksiyonlarından izole edilen en yaygın mantar türleri olduğu bildirilmiştir (12,13) . Bu sorunu aşabilmek için yürütülen çalışmalarda, özellikle amfoterisin B ve ayrıca vorikonazolün transplante edilecek dokuların Candida türleri ile kontaminasyonuna karşı soğuk saklama koşullarında (+4 ve +8°C'lerde 24-72 saat) koruyucu etkinlik gösterdiği bildirilmiştir (14,15) . C. parapsilosis'in de dâhil olduğu bazı Candida türleri ve bir grup maya türü soğuğa adapte olmuş mayalar ("cold-adapted yeast") olarak tanımlanmakta ve soğuk saklama koşullarında süt ve süt ürünlerinde bozulmalara neden oldukları bildirilmektedir (16) .…”