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Effects of Multipurpose Solutions on the Viability and Encystment of Acanthamoeba Determined by Flow Cytometry

Imayasu, Masaki Ph.D.; Tchedre, Kissaou T. Ph.D.; Cavanagh, H. Dwight M.D., Ph.D.

doi: 10.1097/ICL.0b013e31828af147

Purpose: To evaluate simultaneously the effects of multipurpose contact lens care solution (MPS) on the viability and encystment of Acanthamoeba using flow cytometry.

Methods: Viability and encystment rate were evaluated using Acanthamoeba castellanii (ATCC 50514 and ATCC 50370) and three clinical strains of Acanthamoeba spp. isolated from patients with Acanthamoeba keratitis. Acanthamoeba trophozoites (1.0 × 105 cells/mL) were exposed to four kinds of commercially available MPSs for 24 hours. After dispensing the cell suspension into two portions, one portion was stained with 0.004% Congo Red (CR), a fluorescence dye to stain the inner cell wall of cysts, and the other portion was stained with a mixture of Congo Red and 3% sarkosyl (CRS), a detergent to lyse the trophozoites and pseudocysts. Flow cytometric analysis of the treated portions was then carried out on an EPICS ALTRA flow cytometer. The encystment rate and disinfecting efficacies (percentage of rounded trophozoites, “pseudocyst”) were calculated by the rates of CR-stained, CR-nonstained, and CRS-stained populations, respectively. Ultrastructural features of resistant (mature or immature) cysts and pseudocysts were observed by transmission electron microscopy.

Results: Resistant cysts and rounded trophozoites (pseudocysts) were stained with CR, whereas native (unrounded) trophozoites were not. Resistant cysts were also stained with CRS unlike pseudocysts. Three clinical isolates showed higher resistance and higher encystment rates than two ATCC strains when treated with encystment-positive control solution. Disinfecting efficacy of each MPS was not directly related to each encystment rate. Transmission electron microscopy observations showed basic differences in the ultrastructure of pseudocysts produced by MPSs and resistant cysts.

Conclusions: These results suggest that viability and encystment of Acanthamoeba are independent phenomena, and therefore disinfecting efficacy of MPS and encystment rates of Acanthamoeba should be evaluated, respectively. Thus, it is important to evaluate simultaneously the disinfecting efficacies and encystment rates of newly developed premarket MPS using the authors’ novel flow cytometric methods.

Menicon Co., Ltd. (M.I., K.T.T), Kasugai, Aichi, Japan; and the Department of Ophthalmology (H.D.C.), The University of Texas Southwestern Medical Center, Dallas, TX.

Address correspondence and reprint requests to Masaki. Imayasu, Ph.D., Menicon Co., Ltd., 5-1-10, Takamoridai, Kasugai, Aichi, 487-0032, Japan; e-mail:

The authors have no funding or conflicts of interest to disclose.

Accepted February 6, 2013

© 2013 Contact Lens Association of Ophthalmologists, Inc.