| Infectious keratitis is one of the common clinical ophthalmologic diseases leading to blindness. The main pathogenic microorganisms are bacteria, viruses and fungi. The majority of the bacterial and viral keratitis infections are susceptible to anti-infective drugs, with higher cure rate. Fungi, on the other hand, have thick cell wall that are resistant to drug treatment. Treating fungal keratitis with anti-infective drugs is therefore much less effective. Following unsuccessful drug therapy on fungal keratitis and other infectious keratitis, corneal transplant is often carried out to contain the infection and save visual function. Due to the limited supply of corneal, patients often suffer severe damage to the eye sight leading to atrophy or even enucleating whilst waiting for replacement corneal. In recent years, Riboflavin is a vitamin B2, an essential vitamin needed by the human eyes. When riboflavin is exposed to UV light of a specific wavelength (365nm or370nm) in the process of corneal cross-linking, riboflavin can absorb the energy and be excited to generate oxygen free radicals and singlet oxygen. As a result, it fractures and breaks down the nucleic acid, preventing microbial DNA from replication. This mechanism has led to interest amongst the researchers to apply Riboflavin cross-linking to the treatment of drug resistant corneal infections like refractory fungal keratitis. The outcome is expected to not only kill the pathogenic microorganisms, but also increase the corneal hardness and reduce scarring through cross-linking of collagen fibers.ObjectiveTo explore the effectiveness of in vitro standard riboflavin corneal cross-linking to inactivate Fusarium solani and Candida albicans of different concentrations and to treat the fungal keratitis of two groups of New Zealand white rabbits.Methodsvitro test:Fusarium solani and Candida albicans were prepared by separation and purification and diluted with sterile phosphate buffer to produce suspension, using McFarland turbidity adjusted concentration (Fusarium solani suspension1.0Maxwell turbidity units equivalent to3×108CFU/ml, Candida albicans suspension turbidity equivalent to0.5McFarland units1.5×108CFU/ml) with sterile phosphate buffer solution. Two fungal suspensions were diluted10-fold to obtain the stock suspension. The actual concentration of stock suspension was determined with plate colony counting method. We divided our experiment into4groups:control group, riboflavin group, ultraviolet group, corneal cross-linking group (riboflavin+UV). A small amount of liquid dispensed PBS solution and riboflavin were subsequently added to Riboflavin group and corneal cross-linking group so that the density level of the fungus suspension were approximately106,105,104,103,102CFU/ml and the final riboflavin concentration was0.1%. A small amount of liquid dispensed PBS solution are also added to control group and ultraviolet group to achieve density level of the suspension of106,105,104,103,102CFU/ml, but without any Riboflavin. Each group of suspension was prepared within thirty minutes before the experiment began. Corneal cross-linking group and UV groups were placed upon clean desk in standard darkness and exposed to ultraviolet irradiation of cross-linking parameters. Riboflavin group and control groups were placed on the clean desk in the dark without UV irradiation. Duration of each experiment was30min. Concentration level of residual cell (CFU/ml), and inactivation ratios were then calculated and compared for each of the four groups using a flat colony counting method.2Animal models:We produced Fusarium solani and Candida albicans pathogenic fungal keratitis models by corneal stroma injection to New Zealand white rabbits. Each model has20rabbits. Each rabbit has one random eye being treated with Riboflavin cross-linking. Fusarium solani rabbits were treated contralateral with natamycin eye drops and Candida albicans rabbits were treated contralateral with ketoconazole eye drops. By slitlamp microscope,2%sodium fluorescein staining was examined on the day before the treatment and day1,3,7after treatment. Measurement was taken on the maximum diameter of the corneal ulcers and anterior chamber reaction was observed. On the7th and the14th days, the number of eyes where ulcers have healed was recorded for crosslinking r group and drug group. On the14th day the rabbits with unhealed ulcers in cross linking group and drug group were killed and a central corneal of9mm diameter was harvested from each rabbit and weighed immediately. Colonies were counted after72hours of culture. Eyes were also harvested from rabbits of cross-linking and drug group with healed corneal ulcers to perform the corneal histopathology examination.Results1.Riboflavin corneal cross-linking has a moderate effect on the viability of Candida albicans and Fusarium solani in vitro. For Fusarium solani suspension of104,103,102concentration, the residual cell count in the corneal cross-linking group was lower than the control group, riboflavin and ultraviolet group group (P<0.01), the residual difference between the number of cell in the control group, riboflavin and ultraviolet group is statistically insignificant. Inactivation ratio of corneal cross-linking group was25.7%,28.2%,45.1%, respectively for suspension concentration levels of104,103,102Inactivation ratio and the concentration of the suspension fits into a liner regression equation that can be expressed as the following:(Inactivation ratio(per cent))=66.060-9.567(log-transformed cell concentration (Lg CFU/ml))(P<0.01)For Candida albicans with various dilution levels of suspension, the residual cell count of corneal cross-linking group was lower than that of control group, riboflavin group and ultraviolet group group (P<0.01). The residual difference between the cell count in the control group, the group riboflavin and ultraviolet groups was not statistically significant. Inactivation ratio of corneal cross-linking group was32.2%,33.1%,49.0%,57.8%,62.3%, respectively for suspension concentration levels of106,105,104,103,102The inactivation ratios and the concentration of the suspension fits into a liner regression equation that can be expressed as the following:(Inactivation ratio (per cent))=82.043-8.452(log-transformed cell concentration (Lg CFU/ml))(P<0.0001)2.For Fusarium solani group treated with corneal cross-linking, ulcer diameter was materially reduced on the7th day after the treatment. Drug treatment also produced significant reduction in the ulcer diameter7th day after the treatment.25eyes healed in the corneal cross-linking group and13eyes healed in the drug group. We consider the difference in healing rate statistically significant. On the14th day after treatment, cross linking group had17eyes healed and drug group had13healed. We consider the difference in healing rate statistically significant. On the14th day after treatment, the cross linking group had5unhealed ulcers, with Fusarium solani content level of34.74±8.17CFU/g. Drug group on the other hand had7unhealed ulcers, with Fusarium solani content level of54.27±7.35CFU/g. The difference between the two groups was statistically significant.For Candida albicans group treated with corneal cross-linking, ulcer diameter was materially reduced on the7th day after the treatment. Drug treatment also produced significant reduction in the ulcer diameter on the7th day after the treatment. On the7th day,16eye ulcers healed in the cross linking group and12eyes healed in the drug group. We consider the difference in healing rate statistically significant. On the14th day after treatment, cross linking group had17eyes healed and drug group had14healed. We consider the difference in healing rate statistically significant. On the14th day after treatment, the cross linking group had3unhealed ulcers, with Candida albicans content level of28.63±7.79CFU/g. Drug group on the other hand had6unhealed ulcers, with Candida albicans content level of44.15±6.48CFU/g. The difference was statistically significant. Tissue biopsy of corneal showed neat and tight collagen fibers for both groups, with no obvious difference in tissue scarring.Conclusions:1. In vitro, riboflavin corneal cross-linking therapy of standard parameters can partially kill Fusarium solani and Candida albicans of low concentrations. There is a negative correlation between inactivation ratio and log value of the concentration of suspension.2. Riboflavin corneal cross-linking therapy of standard parameters is effective in treating Fusarium solani and Candida albicans keratitis in the New Zealand rabbit model. It also helped repairing corneal tissue. We believe Riboflavin corneal cross-linking therapy has a bigger role to play in the clinical treatment of infectious fungal keratitis. |
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