Nanoemulsions Containing a Coumarin-Rich Extract from Pterocaulon balansae (Asteraceae) for the Treatment of Ocular Acanthamoeba Keratitis.

This study describes the incorporation of a coumarin-rich extract from Pterocaulon balansae into nanoemulsions intended for the local treatment of ocular keratitis caused by Acanthamoeba. The n-hexane dewaxed extract of P. balansae was characterized by HPLC/PDA and UPLC/MS. The presence of four major coumarins was detected, where 5-methoxy-6,7-methylenedioxycoumarin was selected as a chemical marker. This extract was then incorporated into nanoemulsions composed of medium chain triglycerides and egg-lecithin, through spontaneous emulsification. Such a procedure yielded the formation of monodisperse nanoemulsions in a sub-300-nm range, regardless of the amount of extract incorporated (1.0-5.0 mg/mL). The amoebicidal activity against Acanthamoeba castellanii was both dose-dependent and incubation time-dependent. A reduction of 95% of trophozoite viability was detected after 24 h of incubation with a nanoemulsion at 1.25 mg/mL of coumarins, being a similar effect detected for chlorhexidine. These results suggest a potential of the formulations developed in this study as a new strategy for the treatment of ocular keratitis caused by Acanthamoeba.

Genotypic characterization and assessment of infectivity of human waterborne pathogens recovered from oysters and estuarine waters in Brazil.

Waterborne, food-borne and sewage-borne pathogens are a major global concern, with the annual recurrence, most notably during the summer, of outbreaks of gastroenteritis of unconfirmed etiology associated with recreational activities in marine environments. The consumption of contaminated water-based foodstuffs is also related to outbreaks of human illness. The main goals of the present study were: i) to identify the genetic assemblages of Giardia duodenalis cysts in growing and depurated oysters destined for human consumption on the southern coast of São Paulo, Brazil; ii) to verify the main circulating G. duodenalis assemblages and their subtypes in different brackish waters used for the production of mollusks and for recreational purposes; iii) to track the contamination of growing and depurated oysters by the human adenovirus and identify the infectivity of adenoviral particles recovered from oysters before and after depuration; iv) to evaluate the occurrence and genotype of the free-living amoebae of the genus Acanthamoeba in brackish water and oysters from all the sites described above. Four sampling sites in the Cananeia estuary were selected to search for pathogenic and amphizoic protozoa (Giardia and Acanthamoeba respectively): site 1: oyster growth, site 2: catchment water (before UV depuration procedure), site 3: filter backwash (filtration stage of water treatment) and site 4: oyster depuration tank. Oysters at sites 1 and 4 were evaluated for the presence of adenovirus (HAdV). Analysis consisted of conventional microbiological as well as molecular methods. Giardia duodenalis were detected in all the water sites analyzed and the molecular analysis revealed that sub-assemblage AII was the most frequently distributed throughout the estuarine environment, although one sample was identified as belonging to the assemblage C. Acanthamoeba were also isolated from different locations of the estuarine area, and were detected at all the analyzed sites. The majority of isolates belonged to the T3 genotype, while the T4 genotype was identified once. The sequencing reaction of Giardia duodenalis revealed the contamination of three batches of depurated oysters by the sub-assemblage AII. With respect to viruses, seven batches of oysters (four growing and three depurated) were found to be harboring infectious HAdV particles when submitted to plaque assay. Overall, the results of the sequencing reactions combined with the plaque assay revealed that the isolates of Giardia duodenalis and the infectious HAdV particles identified in oyster tissues have the potential to infect humans and pose a threat if consumed raw or lightly cooked. This is the first report on the sub-assemblage AII identified in oysters which are submitted to a cleaning and disinfection procedure prior to human consumption in Brazil. Acanthamoeba specific genotypes were also identified for the first time in a recreational estuarine area in Brazil, contributing to knowledge of their molecular and environmental epidemiology, which is considered scarce even in marine and estuarine areas of the world.

In vitro evaluation of the amebicidal activity of Pterocaulon polystachyum (Asteraceae) against trophozoites of Acanthamoeba castellanii.

The crude extract and hexane, dichloromethane, and methanol fractions obtained from the aerial parts of Pterocaulon polystachyum (Asteraceae) were assayed against Acanthamoeba castellanii, a free-living ameba that causes acute amebic keratitis. Because of its capacity to form cysts, some strains of this protozoan are excellent opportunists and therapy resistant, necessitating a search for new drugs in order to develop more dynamic therapies that make it easier for patients to maintain long-term treatment. In this context, plants with medicinal properties have been analyzed. The broad-spectrum activity against a range of pathogenic fungi shown by extracts of P. polystachyum, together with the use of antifungal drugs as antiprotozoals, made it important to evaluate the amebicidal activity of these plant extracts against A. castellanii. The greatest activity was observed in the treatment with the hexane fraction, which lysed approximately 66% and 70% of the trophozoites in 48 and 72 h, respectively, preventing encystment.