Microbial decontamination of human donor eyes with povidone-iodine: penetration, toxicity, and effectiveness

Abstract

Background/aims: Povidone-iodine (PVP -I) is applied for microbial decontamination of human eyes donated for transplantation. Concentrations and immersion times vary greatly. The effectiveness and toxicity of PVP-I were assessed for different decontamination protocols.

Methods: Human donor eyes and corneas were immersed in different concentrations (5-100 mg/ml) of PVP-I for different times (2-30 minutes). The penetration of iodine into the corneal tissue was assessed by x ray microanalysis. Microbial contamination was determined by taking cultures of the limbal areas and storage solutions and by incubation of the corneoscleral buttons in antibiotic-free culture medium. Cytotoxicity of PVP-I for corneal fibroblasts in culture was assessed using the MTT assay.

Results: Depending on concentration and immersion time iodine was found to penetrate into the epithelium, Bowman's layer, and stroma in amounts equivalent to 2-40 mg/ml PVP-I. The MTT assay demonstrated that 2.5 mg/ml PVP-I caused total damage to fibroblasts in vitro. Rinsing eyes with tap water and subsequent immersion in PVP-I reduced the rate of contamination from 82 out of 106 to 69 out of 106 and 37 out of 106, respectively. Antibiotics in the storage medium further reduced contamination from about 40% to 3%. Microbial contamination was not reduced by increasing the concentration and immersion times beyond 5 mg/ml PVP-I for 2 minutes.

Conclusion: Immersion of human donor eyes in 5 mg/ml PVP-I solution for 2 minutes significantly reduces microbial contamination of donor corneas without relevant penetration of iodine into the corneal layers. Higher PVP-I concentrations and longer immersion times do not further reduce contamination, whereas the amount of iodine penetrating the corneal layers is elevated above the level cytotoxic for corneal fibroblasts. In view of this, concentrations above 5 mg/ml of PVP-I and immersion periods over 2 minutes are not recommended for reduction of the contamination rate of donor eyes.

Figure 1

Scanning electron micrograph of the transverse side of a corneal piece. Epithelium (Ep), Bowman's layer (B), stroma (S), Descemet's membrane (D), and endothelium (En). Arrowheads indicate the sites of x ray microanalysis.

Figure 2

PVP-I concentration and P/B ratio of iodine for standard samples (see Methods) showing the significant correlations for high protein (R=0.98) and low protein (R=0.99) standards. P/B ratios for iodine expressed as mean (SEM) of values assessed in three experiments.

Figure 3

EDAX spectra of a cornea immersed in 50 mg/ml PVP-I for 10 minutes analysed in the basal layer of the epithelium (A) and anterior stroma (B). For details see text.

Figure 4

Penetration of iodine in the epithelium (A), Bowman's layer (B), anterior stroma (C), and mid-stroma (D) of the cornea expressed as P/B ratio for iodine after immersion in 5, 20, 50, 100 mg/ml PVP-I solution for different time periods up to 30 minutes. Results are representative of five experiments.

Figure 5

Dose/response curve of PVP-I solutions for human corneal fibroblasts. Damage was assessed with the MTT assay directly after exposure of the cells to the PVP-I and 48 hours later. Mean (SD) of the means obtained in five experiments in eightfold. Asterisks indicate damage significantly different from controls.