Original article
Evaluation of In Vitro Efficacy of Combined Riboflavin and Ultraviolet A for Acanthamoeba Isolates

https://doi.org/10.1016/j.ajo.2011.07.025Get rights and content

Purpose

To evaluate in vitro the amoebicidal effects of riboflavin and ultraviolet A (UVA) collagen cross-linking.

Design

Experimental study, laboratory investigation.

Methods

Two different strains of Acanthamoeba species were tested identically. Four treatment groups were considered: group 1 consisted of 0.1% riboflavin and 30-minute UVA irradiation; group 2 consisted of 0.1% riboflavin and 60-minute UVA irradiation; group 3 consisted of no riboflavin and no UVA exposure; group 4 consisted of 0.1% riboflavin and no UVA exposure. The application of UVA was performed under the parameters used for in vivo corneal collagen cross-linking.

Results

In all cases, cysts and trophozoites were detected 24 hours after treatment at a radial distance from the center of the seeding point more than 5 mm, indicating that the amoebae were viable. All treated and untreated groups of amoebae from the 2 strains exhibited growth (radii of 14 to 15 mm in groups 1, 3, and 4; radius of 12 mm in group 2). The final morphologic features of the 2 strains of trophozoites that received treatment were similar to those of the initial seeding group and the untreated control group.

Conclusions

The results obtained in our study show that a single dose (30 or 60 minutes) of cross-linking cannot achieve eradication in the 2 different Acanthamoeba strains examined. However, in vitro results do not always indicate in vivo efficacy, so future studies should test the validity of this treatment for Acanthamoeba keratitis.

Section snippets

Amoebic Isolates

The following strains of Acanthamoeba species were used as amebic isolates: Acanthamoeba species 65 was used as an environmental amoeba isolated from superficial water, and Acanthamoeba species 7376 was used as a clinical strain isolated via corneal scraping from a patient with keratitis. The 2 isolates were identified through the microscopic examination of their trophozoites and cysts (Figure 1). The keys reported by Poussard and Pons and by Page were used.19, 20 To confirm the identification

Results

In all groups and for both isolates studied (environmental and pathogenic strains), cysts and trophozoites were detected at distances from the center of the point of application of more than 5 mm after incubation at 30 C in the first evaluation (24 hours), so no re-evaluation or reculture was necessary because of the early growth. This indicated the viability of the amoebae tested. Trophozoites were found at a similar distance from the center of the point of application for all treatment groups

Discussion

In 2008, Iseli and associates used cross-linking to treat 5 patients with infectious keratitis associated with corneal melting,24 but reported only a bacterial or fungal cause. Corneal melting was halted after cross-linking treatment in all the cases. Recently, Morén and associates and Makdoumi and associates also reported that cross-linking therapy was successful in combating severe keratitis of unknown origin.25, 26

The efficacy of ultraviolet light as a disinfectant in contact lens, water, or

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