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Masaki Imayasu, Miya Nomachi, Kissaou T. Tchedre, H D. Cavanagh; A Rapid Determination for Encystment Rate of Acanthamoeba using Flow Cytometry. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6484.
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The encystment process of Acanthamoeba spp is characterized by trophozoite rounding. It was reported that the incidence of Acanthamoeba keratitis (AK) in the United States was related to one specific multi purpose solution (MPS), COMPLETE MoisturePLUS, that induced the transformation of Acanthamoeba trophozoites into resistant cyst. In this study, we have developed a novel method to rapidly compare the encystment rates of Acanthamoeba treated with MPSs using flow cytometry.
Encystment rates of Acanthamoeba castellanii (AC, ATCC50514) treated with eight commercial MPSs (7 PHMB-based and 1 POLYQUAD-based) were analyzed. For flow cytometry, 1.0×105 trophozoites were exposed to each MPS for 24 hours. After dispensing the cell suspension into two aliquots, one aliquot was stained with 0.004% Congo Red (CR), a fluorescence dye to stain the inner cell wall of cyst, and the other aliquot was stained with a mixture of Congo Red and 10% Sarkosyl (CRS), a detergent to lyse the trophozoites. Flow cytometric analysis of the treated aliquots was carried out on EPICS ALTRA flow cytometer. The encystment rate and disinfecting efficacies (percentage of rounded trophozoites) were calculated by the rates of CR-stained and -non-stained part, and CRS stained part. Encystment rates were also calculated by direct counting under microscopy.
Cysts and rounded trophozoites were stained with CR; however native (unrounded) trophozoites were not. Only cysts were stained with CRS. There was a high correlation between the encystment rates obtained by the flow cytometry and those by direct microscopy. The encystment rates of AC by 24-hour treatment of COMPLETE MoisturePLUS were 53.8% by flow cytometry and 57.3% by microscopy. Encystment rates (flow cytometry) of AC treated with other 7 MPSs were between 0.3% and 13.1%, which were significantly lower than COMPLETE MoisturePLUS.
The encystment rates of Acanthamoeba treated with MPS were rapidly determined by the flow cytometric analysis using the intensities of CR fluorescence with or without treatment of Sarkosyl. Disinfecting efficacies of MPS against Acanthamoeba were also evaluated at the same time. Not only high encystment rate but also low disinfecting efficacies of MPS are thought to be associated with the incidence of AK.
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