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Monica Crary, Rhonda Walters, John Bartell, Manal Gabriel, Jagath Kadurugamuwa, Bradley Catalone; Establishing a standard method for evaluating efficacy against Acanthamoeba. Invest. Ophthalmol. Vis. Sci. 2013;54(15):508.
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To establish a standard method for Multipurpose Contact Lens Solution (MPS) efficacy testing against Acanthamoeba by identifying and reducing variables.
Bacterized and axenic cultures of trophozoites were used to determine if culture methods affected MPS efficacy. Cysts that were prepared by starvation with or without Escherichia coli were tested to determine variability in susceptibility to MPS following bacterization. Additionally, the effect of heterogeneous cultures on MPS efficacy was determined by examining the increasing ratios of cysts to trophozoites in axenic cultures that were harvested between 3-7 days of age. Most Probable Number was used for enumeration at different post-test time points to determine the earliest appropriate read date for both cell types. Other variables such as inoculum concentrations and MPS volumes were also examined.
Current recommendations suggest that bacterization of Acanthamoeba is the preferred culture method when evaluating MPS efficacy. However, residual E. coli decreases the available biocide, thereby resulting in variability correlated with bacterial concentration. Current culture methods are limited in their ability to generate homogeneous populations of trophozoites. Axenic trophozoite cultures can contain cysts which contribute to the observed variability of MPS efficacy. Harvesting of trophozoites at 3 days results in a more homogenous population of trophozoites as compared to harvesting at 5 or 7 days. Another variable that contributes to conflicting results in the literature is the duration of incubation prior to enumeration. These studies clearly demonstrate that the minimum incubation period for trophozoites plates is 7 days, whereas for cysts it is 14 days. Earlier time points result in incorrect enumeration. Other potential sources of variability include surface to volume ratio of organisms and MPS.
The maximization of a homogenous population of trophozoites or cysts is necessary to obtain reproducible results when testing MPS efficacy. Variability of test results is affected by the purity of the target cell type. For maximum accuracy, enumeration of cells should not be performed prior to the minimum incubation period. These identified variables have a significant effect on the reproducibility and the performance of MPS against Acanthamoeba.
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