Genotype diversity, phylogenetic analysis and seasonality of isolates of Acanthamoeba spp. in swimming pools in Kafrelsheikh, Egypt.

Species of Acanthamoeba Volkonsky, 1931 are the commonest among free-living amoebae that are widespread in different water resources but with lacking phylogenetic data. This study aims at detecting molecular prevalence and genetic diversity of Acanthamoeba isolates in Kafrelsheikh Governorate, Egypt. Forty-eight water samples were collected from 12 swimming pools; four samples during each season over one year. Samples were filtered, cultivated on non-nutrient agar plates and examined microscopically. Polymerase chain reaction (PCR) and sequence analysis of positive samples targeting diagnostic fragment 3 (DF3) of the small subunit rRNA gene were done. Cultivation succeeded to detect 14 (29%) positive samples while PCR missed three positive samples. The obtained sequences were phylogenetically analysed. The phylogenetic tree was constructed for them with sequences of reference species from the NCBI database. The identified species were Acanthamoeba castellanii Douglas, 1930 (T4), A. astronyxis (Ray et Hayes, 1954) (T9) and A. hatchetti Sawyer, Visvesvara et Harke, 1977 (T11). The prevalence of species of Acanthamoeba was higher during summer and fall. Therefore, the control of the presence of Acanthmoeba spp. in swimming pools needs immediate, effective and practical measures to prevent and control infection with species of Acanthamoeba.

In vitro amoebicidal effect of Aloe vera ethanol extract and honey against Acanthamoeba spp. cysts.

This study evaluated in vitro effect of different concentrations of Aloe vera (A. vera) ethanol extract and honey against Acanthamoeba spp. cysts in comparison with chlorhexidine (the drug of choice for treatment of Acanthamoeba infection) at different incubation periods. Four different concentrations of the tested agents were used, 100, 200, 400, and 600 µg/ml for A. vera ethanol extract and 25, 50, 100, and 200 µg/ml for honey. Isolated Acanthamoeba spp. cysts from keratitis patients were incubated with different concentrations of the tested agents as well as chlorhexidine 0.02% (drug control) for different incubation periods (24, 48, 72 h). After each incubation period, the effect of A. vera extract and honey against Acanthamoeba cysts was assessed by counting the number of viable cysts, determining the inhibitory percentage and detecting the morphological alternations of treated cysts compared to non-treated and drug controls. Both A. vera ethanol extract and honey showed a concentration and time-dependent effect on the viability of Acanthamoeba cysts. In comparison with chlorhexidine (the drug control), A. vera ethanol extract possessed a potent cysticidal activity at all tested concentrations throughout different incubation periods, except for concentration 100 µg/ml which recorded the lower inhibitory effect. With increasing the dose of A. vera ethanol extract to 200, 400, 600 µg/ml, the recorded inhibitory percentages of Acanthamoeba cysts viability were 82.3%, 92.9% and 97.9% respectively, after 72 h compared to 76.3% of chlorhexidine. Similarly, honey at concentrations of 50-100 µg/ml gave higher inhibitory effect of 59% and 76.7%, respectively compared to chlorhexidine which showed an inhibitory percentage of 55.7% after 24 h. Meanwhile, the lowest tested concentration of honey (25 µg/ml) gave an inhibitory effect by 47.7-67% which was less than that of chlorhexidine throughout different incubation periods. With increasing the dose of honey to 200 µg/ml, the inhibitory effect was 98.9% after 72 h higher than that of chlorhexidine (76.9%). Using a scanning electron microscope, Acanthamoeba cysts treated by A. vera ethanol extract showed alternations in their shapes with flattening, collapsing, and laceration of their walls. Also, treated cysts by honey were highly distorted and difficult to identify because most of them were shrinkage and collapsed to a tiny size. On the other hand, chlorhexidine showed less structural and morphological changes of Acanthamoeba cysts. A. vera ethanol extract and honey had considerable cysticidal effects on Acanthamoeba cysts. They may give promising results for treatment of Acanthamoeba keratitis.