Effect of Optisol Supplementation With 0.255 µg/mL Amphotericin B on Elimination of Yeast at 5°C.

PURPOSE: Fungal infections in lamellar keratoplasty are a growing concern. Optisol-GS does not contain an antifungal agent and supplementation with 0.255 µg/mL Amphotericin B (AmpB) has been considered. This study tested the ability of 0.255 µg/mL AmpB in Optisol-GS to eliminate yeast contamination of corneal tissue. METHODS: Three isolates of Candida albicans, 1 of Candida parapsilosis, and 1 of Candida glabrata were tested in Optisol with and without AmpB. Corneoscleral rims stored at -80°C were thawed and placed in 10 multiwell plates (4 per plate). The rims were inoculated with 4 respective loads of yeast: 0, 10, 10, and 10 colony-forming units in 2 sets of 5 for 5 yeasts. One set was filled with Optisol plus AmpB and the other with Optisol only. All 10 plates were incubated at cold storage (2°C-8°C) for 48 hours. After 48 hours, all corneal rims were placed into 10 mL of yeast extract peptone dextrose medium; a swab culture of each well was plated onto Sabouraud plates; and all plates with the remaining Optisol were incubated at 30°C. Yeast growth was monitored for 10 days. Minimum inhibitory concentration and minimum fungicidal concentration were determined. RESULTS: All corneoscleral specimens were positive regardless of fungal load or presence of AmpB. All controls remained negative. Minimum inhibitory concentrations and minimum fungicidal concentrations were equivalent and ranged between 0.5 and 2.0 µg/mL. CONCLUSIONS: AmpB at a concentration of 0.255 µg/mL in Optisol-GS at cold storage (2°C-8°C) over 48 hours did not eliminate yeast from corneal tissue.

Association Between Fungal Contamination and Eye Bank-Prepared Endothelial Keratoplasty Tissue: Temperature-Dependent Risk Factors and Antifungal Supplementation of Optisol-Gentamicin and Streptomycin.

Importance: Fungal contamination and infection from donor tissues processed for endothelial keratoplasty is a growing concern, prompting analysis of donor tissues after processing. Objective: To determine whether eyebank-processed endothelial keratoplasty tissue is at higher risk of contamination than unprocessed tissue and to model eyebank processing with regard to room temperature exposure on Candida growth in optisol-gentamicin and streptomycin (GS) with and without antifungal supplementation. Design, Setting, and Participants: An examination of the 2013 Eversight Eyebank Study follow-up database for risk factors associated with post-keratoplasty infection identified an increased risk of positive fungal rim culture results in tissue processed for endothelial keratoplasty vs unprocessed tissue. Processing steps at room temperature were hypothesized as a potential risk factor for promotion of fungal growth between these 2 processes. Candida albicans, Candida glabrata, and Candida parapsilosis endophthalmitis isolates were each inoculated into optisol-GS and subjected to 2 different room temperature incubation regimens reflective of current corneal tissue handling protocols. Main Outcomes and Measures: Eversight Eyebank Study outcomes and measures were follow-up inquiries from 6592 corneal transplants. Efficacy study outcomes and measures were fungal colony-forming units from inoculated vials of optisol-GS taken at 2 different processing temperatures. Results: Donor rim culture results were 3 times more likely to be positive for fungi in endothelial keratoplasty-processed eyes (1.14%) than for other uses (0.37%) (difference, 0.77%; 95% CI, 0.17-.1.37) (P = .009). In vitro, increased room temperature incubation of optisol-GS increased growth of Candida species over time. The addition of caspofungin and voriconazole decreased growth of Candida in a species-dependent manner. Conclusions and Relevance: Detectable Candida growth in donor rim cultures, associated with a higher rate of post keratoplasty infection, is seen in endothelial keratoplasty tissue vs other uses at the time of transplantation, likely owing in part to eyebank preparation processes extending the time of tissue warming. Reduced room temperature incubation and the addition of antifungal agents decreased growth of Candida species in optisol-GS and should be further explored to reduce the risk of infection.

Impact of temporary hyperthermia on corneal endothelial cell survival during organ culture preservation.

To evaluate temporary exposure to hyperthermia for its impact on endothelial cell density of porcine corneas in organ culture medium containing dextran with regards to possible negative influences of high temperatures during the storage and transport of corneal grafts. Four groups of central discs (diameter 8 mm) from the corneas of both eyes in 40 pigs were first organ-cultured (MEM with 6% dextran 500) for 24 h at 32°C. Ten corneas were then exposed to 40°C in group 1, to 42°C in group 2, to 44°C in group 3, and to 50°C in group 4 for 12 h each. The paired corneal discs for all groups were not treated, stored at 32°C and served as controls. After further organ culture of all corneas for 48 h at 32°C to allow regenerative processes, corneal endothelium was stained with Alizarin Red S and examined by light microscopy. The endothelial cell densities were determined on three central images using a system for the automatic estimation of morphometric parameters of corneal endothelium. Exposure for 12 h to 40°C as well as to 42°C induced no endothelial cell loss. Statistical analysis showed no significant difference of the endothelial cell density between corneas exposed to 40°C and 42°C and the control corneas (40°C treatment: 4736 ± 426 cells/mm(2) and control: 4762 ± 344 cells/mm(2), p = 0.74; 42°C treatment: 4240 ± 363 cells/mm(2) and control: 4176 ± 448 cells/mm(2), p = 0.40). Exposure to 44°C and 50°C lead to total necrosis of the endothelial cell layer. Exposure of organ cultured porcine corneas in dextran containing medium up to 42°C for 12 h does not compromise the endothelial cell density in a clinically relevant manner. Temperatures above 42°C, as it might be the case during transports from the cornea bank to the ophthalmic surgeon, must be strictly avoided as they damage the endothelial cell layer.

A preliminary descriptive analysis of Corneal Transplant Registry of National Eye Bank in India.

PURPOSE: To describe and analyze the Corneal Transplant Registry of National Eye Bank and also evaluate graft outcomes in India. METHODS: All patients who underwent corneal transplant at our center within six months of setting up of Corneal Transplant Registry and installation of database at National Eye Bank were included in the study. The established database was analyzed for utilization, donor and recipient details and graft outcomes. Outcome was assessed at the end of one year follow up. The influence of various donor and recipient factors affecting outcome were evaluated. Visual outcome was analyzed in terms of shift in visual handicap category. Statistical tests like analysis of variance, Kruskal-Wallis test and Chi square tests were applied for determination of clinical significance wherever required. RESULTS: 326 corneas were received from 168 donors; of these, 234 (71.7%) were utilized for transplantation. Out of 177 patients with adequate (one year) follow up (75.6% patients), optical corneal replacement was performed in106 patients and therapeutic keratoplasty in71. 78% (82/106) patients in the optical group retained clear grafts at the end of follow up. 59.7% (49 of 82) of patients who attained clear grafts belonged to visual disability category 3 or worse pre-operatively. 59.1% of these achieved BCVA of ≥6/60 at the end of follow up; thus shifting up their visual handicap category. Primary graft failure was found to be associated with full thickness keratoplasty and not with lamellar procedures (p<0.05) and occurred in 4.2% patients (5) with optical corneal replacement whereas 7.5% patients (8) developed secondary graft failure. Age of donor (p=0.54), death enucleation time (p>0.05), cause of donor death (p=0.15), type of surgical procedures (p=0.538) and indication for surgery did not have any significant effect on outcome. 76% patients who underwent therapeutic graft achieved elimination of corneal infection. CONCLUSIONS: The development of corneal graft registry established an effective means to evaluate our corneal transplantation services. Outcomes of sight restoring corneal transplants performed were comparable to results of graft registries from developed nations.

Pathologic comparison of asymmetric or sulcus fixation of 3-piece intraocular lenses with square versus round anterior optic edges.

OBJECTIVE: To evaluate the pathologic findings of 3-piece intraocular lenses (IOLs) with asymmetric or sulcus fixation in pseudophakic cadaver eyes, comparing IOLs with square or round edges on the anterior optic surface. DESIGN: Comparative case series with pathology. PARTICIPANTS: A total of 661 pseudophakic cadaver eyes, obtained from eye banks within the United States, implanted with different IOLs. METHODS: Anterior segment scanning of whole eyes with a high-frequency ultrasound system or high-resolution anterior segment magnetic resonance imaging followed by gross examination. Selected eyes were processed for complete histopathologic analysis. MAIN OUTCOME MEASURES: Findings from imaging, gross, and histopathologic evaluation that could be related to out-of-the-bag fixation of the lenses. RESULTS: Of 661 pseudophakic cadaver eyes obtained, 13 had 3-piece hydrophobic acrylic IOLs with anterior and posterior square optic edges, and 14 had 3-piece lenses with anterior round edges (13 silicone lenses and 1 hydrophobic acrylic lens) without symmetric in-the-bag fixation. These 27 selected eyes were processed for complete histopathologic analysis. Gross findings in both groups were composed of IOL decentration and tilt, pigmentary dispersion within the anterior segment and on the IOL surface, and iris transillumination defects. Histopathology of the 14 eyes with 3-piece IOLs with round anterior optic edges showed mild focal disruption of the iris pigmented layer and loop protrusion/erosion in the ciliary sulcus. Additional changes observed in the 13 eyes with square anterior optic edge IOLs included iris changes, such as vacuolization, disruption and loss of the pigmented epithelial layers, iris thinning and atrophy, synechiae, and pigmentary dispersion within the trabecular meshwork. One eye also exhibited initial signs of optic nerve disc cupping. CONCLUSIONS: In this series, pathologic findings were more severe in eyes implanted with 3-piece IOLs with square anterior optic edges, suggesting that IOLs with round anterior edges are more suitable for sulcus fixation. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

[The 2009 performance report of the German cornea banks]. / Leistungsbericht der Deutschen Hornhautbanken 2009.

BACKGROUND: In Germany, human tissue for corneal and amniotic transplantation is supplied by 27 cornea banks. METHODS: The Section for Tissue Transplantation and Biotechnology of the German Ophthalmological Society records the cornea banks' activities by means of an annual questionnaire. RESULTS: In 2009, a total of 4,818 corneal grafts were processed by 21 responding cornea banks, and 57% were deemed suitable for transplantation. This ratio is slightly higher than the European average. In addition, German cornea banks released 1,257 amniotic grafts in 2009. DISCUSSION: German cornea banks are currently facing new regulatory issues due to updated legislation regarding tissue transplantation. Recent updates in European law have limited the cutoff time for postmortem blood sampling to 24 h, and this regulation may lead to a significant reduction in potential donors.

Costs and financing. A cost calculation of an up-to-date eye bank in Germany.

PURPOSE: To estimate the averaged cost of processing a corneal graft for keratoplasty. METHODS: We estimated the total running costs of a German corneal bank for one year. All procurement-related expenses were calculated on the basis of 300 donors per year and a disavowal percentage of 50%. RESULTS: The running costs comprise of personnel (2 physicians, 2 technicians), amortization of equipment, laboratory costs, laboratory consumables, occupancy costs and quality management. Annual expenses total 584000 EUR. This aggregation divided by 300 corneal grafts released for transplantation results in a nominal charge of 1950 EUR per corneal graft. DISCUSSION: The DRG system in Germany (in-patients at a base rate of 1.0) refunds only 850 EUR, leaving a financial gap of 1100 EUR per keratoplasty. This financial burden is currently left over to the eye bank and/or the surgeon.

Femtosecond laser-assisted keratoplasty: laboratory studies in eye bank eyes.

PURPOSE: To optimize the surgical technique for performing femtosecond laser-assisted keratoplasty (FLAK) using the IntraLase FS to cut both recipient and donor cornea buttons in eye bank globes. METHODS: FLAK was performed in six globes and six corneoscleral buttons for each of the following trephination patterns: top hat, mushroom, tongue-groove, and vertical. Manual trephination was performed as control. The wound integrity was tested in incisions closed with 8 sutures, 8 sutures with fibrin adhesive, and 16 sutures by measuring the intraocular pressure required to produce graft-host wound leakage (IOP(L)). Light microscopy (LM) and scanning electron microscopy (SEM) were performed to assess cut surface quality and graft-host interface regularity. RESULTS: Mushroom and top hat FLAK had significantly higher IOP(L) than vertical or manual trephination (p < 0.0001) for wounds closed with 16 sutures. There was no difference in IOP(L) between top hat, mushroom, and tongue-groove FLAK in wounds closed with 8 sutures with fibrin adhesive (p > 0.75). LM and SEM demonstrated cut surfaces with good quality and smooth edges. CONCLUSIONS: These preliminary studies show that FLAK produces precise trephination cuts of superior wound strength and stability to that of manual trephination. Adjuvant fibrin glue may further improve wound integrity.

Evaluation of potential organ culture media for eye banking using human donor corneas.

AIM: To evaluate the ability of different commercially available cell culture solutions to preserve human donor corneas during 3 weeks of "closed system" organ culture at physiological temperature. This screening was performed in an attempt to establish a rational basis for the development of a serum-free organ culture medium for eye banking. METHODS: 72 normal human donor corneas were organ cultured for 21 days at 31 degrees C in eight different test media (nine corneas in each group). The basic culture solutions included: minimal essential medium (MEM), MEM with stabilised L-glutamine, M199, DIF-1000, SFM, F99, and F99 with ascorbic acid, insulin, bFGF, transferrin, selenium, and lipids (termed F99-Sr). All media were supplemented with 2% fetal calf serum (FCS), except for MEM, which was also studied at 8% FCS. The evaluation parameters included: (1) the endothelial cell loss as evaluated using trypan blue staining; (2) the ability of keratocytes and endothelial cells to incorporate tritiated uridine into RNA as evaluated using autoradiography and digital image analysis; (3) the leakage of immunogenic keratan sulphate as assessed using ELISA; and (4) changes in storage medium pH, glucose, and lactate content. RESULTS: SFM induced the lowest endothelial cell loss of 14% (SD 2%) and the highest RNA synthesis rates of all test solutions supplemented with 2% FCS. Corneas stored in SFM also showed the least leakage of keratan sulphate and the highest glucose consumption and lactate production. In five media (MEM with 2% FCS, MEM with stabilised L-glutamine, M199, F99, and F99-Sr), comparable and intermediate potentials for organ culture were observed with endothelial cell loss of 16-19%. By contrast, 29% (4%) of the endothelium was lost after storage in DIF-1000. Interestingly, the use of 8% FCS (in MEM) had a marked protective effect on the endothelium, which showed the highest RNA synthetic activity combined with a cell loss of only 11% (4%), compared with 19% (6%) at 2% FCS (p<0.05). CONCLUSION: Among the present test solutions, SFM appears to be the most prominent candidate for a new corneal organ culture medium and should be further tested and possibly refined to effectively substitute serum addition.

A sensitive method for testing the quality of organ culture media and of individual medium components in a cornea bank.

BACKGROUND: It has been suggested that variations in the quality of organ culture preservation media are responsible for variations in early postoperative graft morphology. Spates of such variations have been observed repeatedly for short periods. This paper reports the results of a series of grafts with low postoperative clearing observed during a period of 6 weeks. Simultaneously, preoperative phase-contrast microscopy evaluation of the corneal endothelium revealed that an unusually large proportion of donor corneae were unsuitable for transplantation. METHODS: The corneal storage media were therefore rigorously screened, paying particular attention to specific components and properties of the medium, including L-glutamine, amphotericin B, water quality, pH, and the glassware used. Possible toxic effects were identified by means of a sensitive growth assay performed using isolated human corneal endothelial cells. RESULTS: The evaluation demonstrated that both the water quality and the L-glutamine which had been used for preparation of the medium were substandard during the period in which poor clinical results were obtained. CONCLUSION: It is recommended that cornea banks undertaking long-term organ culture use standardized protocols and carefully monitored equipment. The quality of the basal media and supplements should be routinely checked.

Ex vivo phosphorus magnetic resonance spectroscopy on eye bank corneas and corneal metabolic health.

BACKGROUND: Since a potential exists for untoward effects on the cornea from the high magnetic fields and radio-frequency energies, and the further manipulation required for phosphorus-31 magnetic resonance spectroscopy (31P-MRS), we determined the effects of this technology on tissues using paired human corneas (n = 4) meeting criteria acceptable for transplantation. METHODS: Slit-lamp biomicroscopy, pachometry, specular microscopy, and redux fluorophotometry were performed on all corneas. One cornea of each pair was examined (< 30 min) by 31P-MRS. Following 31P-MRS, slit-lamp biomicroscopy, pachometry, and redox fluorophotometry were again performed. RESULTS: Data tabulated included the 31P energy modulus (1.37 +/- 0.28), the ATP/Pi (2.92 +/- 0.59) and SP/Pi (0.76 +/- 0.04) ratios, and the intracorneal pH (7.24 +/- 0.09). CONCLUSION: Since there were no significant differences in slit-lamp biomicroscopy, endothelial density and morphometry, cell counts, and pachometric and redox fluorophotometric measurements between corneas of each pair before and after 31P-MRS analysis, it was concluded that there was no detectable metabolic damage secondary to such analysis. This study suggests that MRS analysis of human eye-bank tissues does not damage the cornea metabolically and may provide a practical evaluation of the health of the cornea at the biochemical level.

Physiological state of the rabbit cornea following 4 degrees C moist chamber storage.

Eyes from female albino rabbits (1.9-2.3 kg) were enucleated immediately following euthanasia along with the lids and conjunctiva. The globes were moist chamber-stored (in a 4 degrees C refrigerator with the lids closed and the cornea facing downwards) for 12-500 hr. Central corneal thickness (by ultrasound) increased from 350 to approximately 650 microns within 72 hr but changed little thereafter. In the latter period, the relative fluid pressure of the globe (pneumatonography) dropped to less than 10 mmHg, aqueous bicarbonate (tCO2) levels fell to less than 5 mM and anterior chamber fluid tonicity decreased progressively (especially after 24 hr storage) to reach values of around 200 mosmol l-1 by 7 days. Storma-endothelium preparations of the corneas, after 2 hr of in vitro equilibration with a bicarbonate-Ringer solution (supplemented with glucose, adenosine and glutathione) were evaluated for their ability to undergo deturgescence under silicone oil or to pump fluid against a hydrostatic pressure of 20 cmH2O. Corneal preparations from up to 7 days storage showed rapid (60 to 135 microns hr-1) and complete deturgescence (net change in thickness of 140-180 microns) that was maintained. Thereafter, the ability to show deturgescence declined to zero by 10 days. In marked contrast, endothelial fluid pump activity (of approximately 5 microliters hr-1) was manifest for only 36 hr after which time this function rapidly declined. Most corneas stored for periods longer than 48 hr exhibited a continuous leak (of -1 to -5 microliters hr-1). The results indicate that corneal deturgescence and endothelial fluid pump function are not necessarily coupled in vitro.