Amoebicidal activity of oxygenized riboflavin photoactivation: BPerox-UVA as a novel combination therapy for keratitis

Abstract

Purpose : Acanthamoeba Keratitis available therapies consist of highly toxic chemotherapeutic agents and biocides intensively administered for a long time. Riboflavin (RF) + UVA alone in doses up to 10x higher than recommended do not seem to have either trophocidal or cysticidal effects. The purpose of this work is to test a modified photochemical treatment against Acanthamoeba castellanii.

Methods : Photochemical testing on trophozoites and cysts from A. castellanii (AC) Neff (ATCC 30010) genotype T4. The inoculum was prepared form cultures in non-nutrient agar plates supplemented with heat-killed Escherichia coli. Trophozoites were obtained after incubation for 12 h at 30°C and encystment was spontaneously induced after 2 weeks once the culture was left starving. A cell suspension with a loopful (10 μl) of AC in 500 μl of saline solution was pipetted in 12-well culture plates in combination with 200 μl of BPerox containing RF 0.1% or 0.5% plus hydrogen peroxide 0.004%. Subsequently, this mixture was irradiated at 3 mW/cm² UVA for 30 min or 10 mW/cm² for 10 min, respectively. After the irradiation therapy, the microorganisms were subcultured and incubated at 30°C. By direct microscopic examination, the viability of AC was assesed by visual counts and t-test analyses were performed. Control groups were established. Plates were examined up to 2 weeks prior determination of a negative culture.

Results : Complete trophocidal activity was seen with BPerox 0.1% - 0.004% + UVA for 30 min photoactivation compared to controls (p ≤ 0.001). BPerox phototherapy at the same dose produced a significant cysticidal effect contrasted to controls (p ≤ 0.003). BPerox 0.5% - 0.004% + UVA for 10 min demonstrated 100% reduction of total trophozoites and a significantly cyst reduction compared to controls (p ≤ 0.003 and ≤ 0.014). RF + UVA, hydrogen peroxide + UVA and monotherapy treatments did not display any amoebicidal activity in contrast to the positive control.

Conclusions : The amoebicidal activity of BPerox-UVA photoactivation against living forms of AC in vitro is significant, which is new for this condition. The safety and efficacy of this treatment is currently being evaluated in vivo.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

RF-UVA did not display any amoebicidal activity in contrast to the positive control.

 

The efficacy of BPerox-UVA demonstrated significant killing for both living forms of A. castellanii.