Classificação morfológica, genotipagem e avaliação da patogenicidade de isolados clínicos e ambientais de Acanthamoeba em Vitória e região metropolitana (ES)

• Ano de defesa: 2012
• Autor(a) principal: Possamai, Cynara Oliveira
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal do Espírito Santo; BR; Mestrado em Doenças Infecciosas; Centro de Ciências da Saúde; UFES; Programa de Pós-Graduação em Doenças Infecciosas
• Palavras-chave em Português: Acanthamoeba; Isolation; Genotyping; Thermotolerance; Osmotolerance; Isolamento; Genotipagem; Termotolerância; Osmotolerância; Doenças Infecciosas e Parasitárias; 61
• Link de acesso: http://repositorio.ufes.br/handle/10/5969

The genus Acanthamoeba comprises amphizoic protozoa with a wide environment distribution. They can cause serious diseases in humans, such as amoebic keratitis and granulomatous amoebic encephalitis. Thus, the factors involved in the pathogenicity of Acanthamoeba are being investigated as major interests to identify strains able to cause infection. The aim of this study was to investigate the occurrence of Acanthamoeba both in clinical and environmental samples, characterize the isolates by morphological parameters, genotyping and also evaluate the pathogenic potential. Clinical samples were collected from patients with a suspicious diagnosis of amoebic keratitis throughout corneal scrapings. Environmental samples were collected from dust, soil, swimming pool, tap, sea, and flood waters in Vitoria metropolitan regions, Espirito Santo State, Brazil. All samples were cultured on soy agar. Samples from corneal scrapings were also collected in Page saline and subjected to a reaction of semi-nested PCR. Positive cultures for Acanthamoeba, previously identified based on the cysts and trophozoites morphology, were selected, cloned and classified into morphological groups I, II or III. Genotyping of isolates was performed by partially sequencing the 18S rDNA gene while the pathogenic potential of cloned cultures was assessed by thermo and osmotolerance assays. From all samples cultured on agar, 90 were from environmental sources, 16 from corneal scrapings and nine from contact lenses (CL). Of these, 38 (33 from environmental samples, four from clinical samples and one from CL sample) were positive for Acanthamoeba. Among the 38 positive isolates, 28 were successfully cloned (24 from environmental isolates, three from clinical isolates and one from CL isolate). Twenty six cloned samples showed morphological characteristics of group II, one of group I (soil) and one (tap water) could not be classified according to morphological parameters. Four cases of amoebic keratitis could only be confirmed by molecular diagnosis. All clinical, CL and most of the environmental isolates sequenced were classified as T4 genotype. Among the environmental isolates, two were grouped in genotype T11 (pool) and one in T1 (dust). All cloned isolates subjected to the thermotolerance assay grew at 28 ºC and 37 ºC. The same result was observed in osmotolerance tests at 0.1M and 0.5M mannitol. Neverthless, while most of the cloned isolates were able to grow at 1.0M mannitol, none of the isolates were able to grow at 42 ºC. The present study confirms the predominance of morphological group II and genotype T4 in clinical and environmental isolates of Acanthamoeba in Espirito Santo State and first time reports the T1 genotype isolation of Acanthamoeba in Brazil. This work also demonstrates the presence of potentially pathogenic isolates at the environment, including samples of flood and sea waters, which may represent a risk factor for the development of infections caused by Acanthamoeba. Furthermore, the methodology used in this study could be used for a fast, sensitive and specific diagnosis of Acanthamoeba keratitis.