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Shawn C Lynch, Christopher Kovacs, Kimberly A Millard, Suzanne F Groemminger, Timothy W Morris; Influence of Buffering Systems on Acanthamoeba Encystment Following Prolonged Storage with Experimental Formulations and a Novel Hydrogen Peroxide-based Disinfecting Solution. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4645.
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Acanthamoeba protozoa are capable of undergoing a transition from active, mobile trophozoites into a dormant cyst form when exposed to a harsh environment. Previous published work associated this encystment phenomenon with propylene glycol (PG), a demulcent component of a multipurpose disinfecting solution that was recalled in 2007 due to an association to Acanthamoeba keratitis. Here, we examine the effects of PG on encystment of Acanthamoeba in a variety of model systems, including different buffering agents as well as in a novel hydrogen peroxide-based disinfecting solution.
Test articles included phosphate, borate and phosphate/citrate-buffered solutions (P-BS, B-BS and P/C-BS, respectively) with and without PG (1%), as well as a novel 3% hydrogen peroxide-based disinfecting solution with and without PG (1%). Control articles included the hydrogen peroxide-based disinfecting solutions Clear Care and Oxysept. A. castellanii ATCC 50370 trophozoites were cultured in antibiotic-free AC6 medium, treated with test and control solutions for 24 hours, 7 days and 14 days, and assessed for encystment via fluorescent microscopy with Calcofluor White staining.
Encystment rates for P-BS with PG were greater than 57% at 24 hours exposure. PG in combination with all other solutions tested demonstrated encystment rates of less than 1%. Encystment rates for all solutions incubated for 7 and 14 days remained low, at less than 5%.
Encystation of Acanthamoeba is due to overall solution formulation as opposed to just the presence of PG in isolation. The lack of encystment observed after 24 hours exposure in B-BS, P/C-BS, and the novel hydrogen peroxide solution each containing 1% PG, compared to the strong positive signal observed in P-BS containing 1% PG, suggests that a compensatory influence of all components of a solution is required to induce encystment. Additionally, prolonged exposure of trophozoites to the solutions tested, including the novel hydrogen peroxide solution post-neutralization, did not stimulate encystment, further challenging the concept of PG-induced encystment. Taken together, these data highlight that encystment of Acanthamoeba is a complex process influenced by a multitude of factors, rather than just a single component.
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