Mestrado em Doenças Infecciosas
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Navegando Mestrado em Doenças Infecciosas por Assunto "Acanthamoeba"
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- ItemAvaliação da virulência de isolados dos genótipos T3, T4 e T5 de Acanthamoeba provenientes de amostras clínicas e ambientais(Universidade Federal do Espírito Santo, 2018-03-23) Fernandes, Débora da Vitória de Melo; Bueloni, Cinthia Furst Leroy Gomes; Falqueto, Aloísio; Fux, Blima; Leite, Gustavo RochaThe genus Acanthamoeba comprises protozoa that are widely distributed in diverse environments and across all continents and that are capable of causing infections in humans, such as keratitis and granulomatous encephalitis. The pathogenesis of Acanthamoeba constitutes a multifactorial process, with factors that involve both ameba and host, but this mechanism of pathogenicity is not yet fully elucidated. There is little standardization in the methodologies applied here, with variable protocols in the literature. The objective of this work was to identify the virulence variations of six isolates of Acanthamoeba, from clinical and environmental backgrounds, with three different genotypes, T3, T4 and T5 - representing the 20 types of findings, and with two doses of amoebae on three types different from mammalian cell lines, MDCK, VERO and CHO, using cytotoxic effect and cytotoxic effect methodologies. Clinical samples are obtained from cultured corneal scrapings of patients diagnosed with amoebic keratitis, and as environmental samples are obtained from faucet outlet, flood water and dust, collected and axenized between the years 2014 and 2017. Considering that prolonged cultivation of Acanthamoeba isolates may cause a decrease or loss of virulence of the amoeba, we use the passage of the amebae in MDCK cell line to reactivate the virulence of the isolates in prolonged culture. Our data demonstrated that a cell line more susceptible to conditioned medium (cytotoxic effect) was MDCK, followed by VERO and CHO. However, for the cytopathogenicity assay, CHO was one more susceptible to exposure to Acanthamoeba trophozoites, followed VERO and MDCK. In the cytopathic effect, the results varied according to the isolate used, the dose used of trophozoites and the passage in cell line. The increase in the number of amoeba trophozoites in incubation with the cell lines was able to provoke an increase in the virulence of the isolates of the three genotypes in the cytopathic effect test, especially the VERO and CHO lines. In the cytopathic effect test, the most virulent genotype was T5, followed by T4 and T3, the most virulent isolate was A3P4 (T5) of environmental origin. Thus, it is concluded that the results obtained with the cytopathic and cytotoxic effect tests with the different mammalian cell lines have variations related to the type of mammalian cell line used, as well as the inherent characteristics of each isolate. Therefore, the results obtained here may be useful for the planning of future research related to the pathogenicity studies of Acanthamoeba.
- ItemAvaliação do efeito do cultivo axênico prolongado e da interação com células MDCK em propriedades de patogenicidade de um isolado clínico do gênero Acanthamoeba(Universidade Federal do Espírito Santo, 2017-07-21) Prado, Guilherme Pinho do; Bueloni, Cinthia Furst Leroy Gomes; Falqueto, Aloísio; Fux, Bluma; Moreira, Narcisa Imaculada BrantThe free-living amoeba (FLA) of the genus Acanthamoeba are protozoan found in all environments of the world, and can cause diseases such as Keratitis and Granulomatous Encephalitis. Advances do not know the amoebae and their invasive process, their phenotypic and genetic variability, as well as the development of diagnostic and therapeutic methodologies, are only being fulfilled with the microorganisms cultivation tool. However, their prolonged cultivation and their passage into hosts (or cells) can change a cellular machinery and modify mechanisms related to pathogenicity, making them amoebas more or less virulent. In the present work, we investigated in vitro characteristics associated with the pathogenicity of Acanthamoeba castetellanii (Genotype T4), using four samples from a clinical isolate, originating from corneal scraping, in order to investigate its virulence. Epithelial cells of the MDCK lineage lengths for checking the cytopathic effect. It was also evaluated the environment where as amebas were cultured (conditioned medium) to verify the activity of proteases in gel and the cytotoxic effect generated in cultures of MDCK. The percentages of binding and response of the amoeba culture from chlorhexidine exposure were verified. The data obtained in the work confirm the pathogenic potential of the samples, especially those that underwent passage in MDCK. The environment did not produce a significant cytotoxic effect on a cell monolayer. The zimographic profile showed a very similar pattern in all the samples tested, differentiating only one sample from a long period of axenization, with a band of approximately 133 kDa increasing. It was verified that the binding percentage of the newly axenized samples was higher than the development period, and that the latter test obtains higher binding rates after interchange with MDCK. The chlorhexidine test demonstrated that, despite the sample from the same source, as sample maintenance conditions led to different behaviors. In this work was observed in two years of axenic cultivation, an amoeba loses virulence, yet it recovers it after passage in cells, as an MDCK. Collaborate for studies in Acanthamoeba advance demonstrating a plastic amoeba's potential in managing its pathogenic profile.
- ItemClassificação morfológica, genotipagem e avaliação da patogenicidade de isolados clínicos e ambientais de Acanthamoeba em Vitória e região metropolitana (ES)(Universidade Federal do Espírito Santo, 2012-08-28) Possamai, Cynara Oliveira; Pereira, Fausto Edmundo Lima; Bueloni, Cinthia Furst Leroy Gomes; Costa, Adriana Oliveira; Falqueto, AloísioThe 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.