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Karim Makdoumi, Anders Backman, Jes Mortensen, Sven Crafoord; In Vitro Investigation of Riboflavin/UVA-mediated Elimination of Acanthamoeba Castellanii. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6210.
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To assess the antiprotozoal effect of Riboflavin photosensitization by Ultraviolet light A (365 nm).
Suspensions of Acanthamoeba castellanii, (ATCC 30010) were cultured and prepared in peptone-yeast-glucose (PYG) medium to a concentration of 100, 000 protozoa/ml. 10 µl was removed, placed in a well and subsequently mixed with 50 µl PBS plus riboflavin to achieve an end concentration of 0.01 %. 10 µl was removed for culturing in 140 µl PYG medium. The remaining 50 µl was exposed for UVA light with a dose of 5.475 J/cm2, 10 ul was cultured i PYG and the illumination period was repeated. After concluding the second UVA exposure 10 µl was removed and cultured in the same medium as specified previously. For each solution a negative, UVA-non-exposed solution was prepared with origin from the same source suspension. Illumination and culturing took place in a dark room to avoid photodegradation of riboflavin caused by background light. Growth of the protozoa continued for one week. The experiment was repeated three times in full.
The elimination of Acanthamoeba was achieved by the exposure to UVA-riboflavin, with a more pronounced reduction of protozoa at the higher UVA-dosages, consistent with a dose-response relationship regarding UV light. The non-exposed suspensions had a slightly lower microbial count than source solutions. Addition of riboflavin did not potentiate the elimination of protozoa. The count of microorganisms on the final day of experiments was equivalent to a reduction of approximately 50 % at the higher UV exposure dose. A statistically significant UVA-mediated reduction could be confirmed at these levels (p<0.05).
Neither riboflavin solitarily nor the photosensitization of the vitamin mediate harm to undamaged Acanthamoeba trophozoites, however, ultraviolet light A (at 365 m) caused elimination of the protozoa, with a dose-response relationship. If considered as an future keratitis therapy, the in vivo effect of vitamin B2 photoactivation may differ from the experimental outcome, as could the result of amoebicidal drugs combined with the process.
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