We have developed and optimized a 96-well microtiter plate assay, based on the reduction of alamarBlue, to assess the efficacies of much needed new antimicrobials against Acanthamoeba species. This assay has been optimized for determination of drug efficacy against two potentially pathogenic species, Acanthamoeba castellanii and Acanthamoeba polyphaga, and has been validated by comparison of their relative susceptibilities to chlorhexidine, a drug widely used to treat Acanthamoeba keratitis. The results demonstrate that the assay is comparable to a manual counting assay and that A. polyphaga is more resistant to chlorhexidine than A. castellanii. Thus, by use of the manual counting assay, 3.125 M chlorohexidine was almost completely effective against A. castellanii, whereas this concentration was less than 20% effective against A. polyphaga. Similar results were obtained by the alamarBlue assay. The new assay was used to determine the relative susceptibilities of A. castellanii and A. polyphaga to the alkylphosphocholines (APCs) hexadecylphosphocholine (hexadecyl-PC; miltefosine) and octadecylphosphocholine (octadecyl-PC) as well as an alkylgycerolphosphocholine, edelfosine. Both APCs studied were equally effective against A. castellanii, but octadecyl-PC was less effective than hexadecyl-PC against A. polyphaga. Both APCs were more effective than edelfosine against both Acanthamoeba species. A. polyphaga was found to be significantly less susceptible to each of the phosphocholine analogues. The newly described assay offers a number of advantages over those described previously. It is less labor-intensive than previously described assays and is sensitive and rapid, and the results can be read in a nonsubjective manner. As it is based on a standard 96-well, microtiter plate, it is amenable to automation and high throughput.
Objectives: Previous studies have reported the ability of several phospholipid analogues to successfully inhibit the growth of Acanthamoeba species in vitro. This study tests further phospholipid analogues, either as free drug or in liposomal formulations, and unlike previous studies, examines their comparative toxicities to mammalian cells.
Methods:The relative cytotoxic activities of the phospholipid derivatives hexadecyl-PC, octadecyl-PC, elaidyl-PC, erucyl-PC and edelfosine, against Acanthamoeba castellanii, Acanthamoeba polyphaga and a rabbit corneal epithelial (RCE) cell line, was determined by the alamarBlue TM assay. Free and liposomal formulations were compared for hexadecyl-PC and elaidyl-PC.Results: Both hexadecyl-PC and octadecyl-PC (IC 50 values between 3.9 and 7.8 mM) demonstrated considerable activity against A. castellanii, as did elaidyl-PC (IC 50 values between 15.6 and 31.25 mM). Both hexadecyl-PC and elaidyl-PC also proved effective against A. polyphaga (IC 50 values between 15.6 and 31.25 and between 31.25 and 62.5 mM, respectively). In contrast, neither erucyl-PC nor edelfosine was inhibitory against either Acanthamoeba species. The growth of RCE cells was inhibited by octadecyl-PC, erucyl-PC and edelfosine (octadecyl-PC and erucyl-PC IC 50 values between 7.8 and 15.6 mM and edelfosine IC 50 values between 31.25 and 62.5 mM). Liposomal formulations of hexadecyl-PC and elaidyl-PC were less effective than free drug against both Acanthamoeba species.Conclusions: These results demonstrate that hexadecyl-PC has the highest therapeutic index and is the most promising for the treatment of acanthamoebiasis.
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