In vitro comparative assessment of different viability assays in Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites.

The species of the genus Acanthamoeba are opportunistic protozoan parasites that cause different diseases in humans, such as amoebic keratitis and granulomatous encephalitis. The rise in the rate of Acanthamoeba keratitis, mainly due to the increase in contact lens wearers, turns the development of viability assays using a multi-well plate reader as a tool for screening new antiamoebic agents in vitro into an important goal. In our study, the viability assays PrestoBlue®, resazurin sodium salt, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and CellTiter96® were tested for their suitability as time-saving alternatives to the classical manual or direct-counting method, assessing the effect of the antiamoebic agent chlorhexidine digluconate and temperature on Acanthamoeba castellanii (ATCC® 30234™) and Acanthamoeba polyphaga 2961. Although resazurin and MTT have already been previously used in amoeba viability assays to test the activities of antiamoebic agents in vitro, it is the first time that PrestoBlue® and CellTiter96® are used for this purpose. Results indicated that the viability assays were strain-dependent leading in some cases to an overestimation of the real situation of viable cells. This implies that each viability assay ought to be set up for each amoeba strain studied.

Assessment of the efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and Tetrahymena spp.

The efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and two Tetrahymena spp. was determined based on the European Standard EN 1276:2009 suspension test. Trophozoite viability was assessed by determination of the membrane integrity using flow cytometry as a fast screening technique. Bovine serum albumin was added to simulate clean (0.3 g/liter) and dirty (3 g/liter) conditions. Benzalkonium chloride caused cell lysis at concentrations above 50 mg/liter under clean and dirty conditions. A concentration of 50 mg of free chlorine per liter had a strong biocidal effect on acanthamoebae and tetrahymenae after 15 min under clean and dirty conditions. Our results suggest that benzalkonium chloride and sodium hypochlorite were effective against the three microorganisms at concentrations commonly applied in the food industry.

Development of colorimetric microtiter plate assay for assessment of antimicrobials against Acanthamoeba.

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 microM 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.

Biofilms, thermophilic amoebae and Legionella pneumophila--a quantitative risk assessment for distributed water.

A simplistic quantitative microbial risk assessment (QMRA) based on the maximum risk curve (r = 1) was developed for Legionella within a water distribution system. Both biofilms and a thermophilic isolate of acanthamoebae were shown to increase the resistance of Legionella to conventional thermal disinfection by between one and two logs respectively. The level of risk presented to consumers was shown to exceed the USEPA 10(-4) benchmark in many cases tested. This was caused, in part, by the sensitivity of the risk model but also through a lack of reliable dose-response data for Legionella. Not withstanding this, the current study provided comparative information on the efficacy of conventional disinfection against Legionella. Combined chlorine was shown to reduce the risk of infection by as much as 1-log when compared to free chlorine, although thermal disinfection provided the most effective means of risk reduction. Biofilm detachment and the interaction of Legionella with acanthamoebae were two important ecological factors that significantly increased the risk of legionellosis, and thus should be further considered in the refinement of QMRA models.

In vitro assessment of susceptibility of Acanthamoeba polyphaga to drugs using combined methods of dye-binding assay and uptake of radiolabelled adenosine.

A technique based on binding of eosin dye to cell was applied to quantitate Acanthamoeba trophozoites in culture. Using this technique in combination with the uptake of radiolabelled adenosine, we assessed the activities of triazole (saperconazole), imidazole (ketoconazole, miconazole) and diamidine (berenil, pentamidine, dibromopropamidine) compounds against A. polyphaga trophozoites. The quantity of dye bound to the trophozoites correlated (r = 0.99) with the number of organisms per well. When inhibition of the growth of Acanthamoeba was the only parameter used to measure the effectiveness of these drugs, saperconazole was found to be most active with IC50 (concentration of drug that inhibited by 50%, the growth of A. polyphaga trophozoites incubated at 28 degrees C for 48 h) of 0.95 microM. The IC50s of the other drugs ranged from > or = 1500 microM for micronazole, the least active, to 3.0 microM for berenil. However, when efficacy was assessed by combining inhibition of growth with the uptake of [14C-8]adenosine by the drug treated organism, the diamidines, particularly berenil and pentamidine were considered most potent.