Menicon Co., Ltd., Kasugai, Aichi, Japan.
Eye Contact Lens. 2013 May;39(3):228-33. doi: 10.1097/ICL.0b013e31828af147.
To evaluate simultaneously the effects of multipurpose contact lens care solution (MPS) on the viability and encystment of Acanthamoeba using flow cytometry.
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 × 10(5) 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.
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.
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.
使用流式细胞术同时评估多用途隐形眼镜护理液（MPS）对棘阿米巴的生存能力和囊包形成的影响。
使用棘阿米巴Castellanii（ATCC 50514 和 ATCC 50370）和从棘阿米巴角膜炎患者中分离出的三种临床株棘阿米巴属来评估生存能力和囊包形成率。将棘阿米巴滋养体（1.0×10(5)个细胞/mL）暴露于四种市售的 MPS 中 24 小时。将细胞悬液分至两部分后，一部分用 0.004％刚果红（CR）染色，这是一种荧光染料，可染色囊包的内壁，另一部分用刚果红和 3％Sarkosyl（CRS）的混合物染色，这是一种可裂解滋养体和假囊包的去污剂。然后在 EPICS ALTRA 流式细胞仪上对处理部分进行流式细胞术分析。通过 CR 染色、CR 非染色和 CRS 染色群体的比率分别计算囊包形成率和消毒功效（圆形滋养体的百分比，“假囊包”）。通过透射电子显微镜观察抗药性（成熟或不成熟）囊包和假囊包的超微结构特征。
抗药性囊包和圆形滋养体（假囊包）用 CR 染色，而天然（未圆形）滋养体则未染色。与假囊包不同，抗性囊包也用 CRS 染色。与 ATCC 株相比，三种临床分离株在使用囊包阳性对照溶液处理时表现出更高的抗性和更高的囊包形成率。每种 MPS 的消毒功效与每个囊包形成率并不直接相关。透射电子显微镜观察显示，MPS 产生的假囊包和抗性囊包的超微结构存在基本差异。
这些结果表明，棘阿米巴的生存能力和囊包形成是独立的现象，因此应分别评估 MPS 的消毒功效和棘阿米巴的囊包形成率。因此，使用作者的新型流式细胞术方法同时评估新开发的上市前 MPS 的消毒功效和囊包形成率非常重要。
