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Free living amoebae as causative agents of human infections.
Markovičová, Ivana ; Nohýnková, Eva (advisor) ; Doležal, Pavel (referee)
Free living amoebae as causative agents of human infections are world wide occuring organisms with the ability of dual lifestyle both in nature, which is their original habitat, so within host tissue. The aim of this theses is to summarize the basic available knowledge of the pathogenic species Naegleria fowleri and Balamuthia mandrillaris and of the pathogenic genus Acanthamoeba which invade CNS and are causative agents of rare but serious human infections, often leading to death of a patient. The exception of their deathly consequences is an Acanthamoeba keratitis infection in which non curing cases lead to a significant visual impairment and even to visual loss. In each of the chapters, the work is tend to summarize transmision ability, mechanisms of pathogenesis and pathophysiology of these three free living amoebae in order to describe their ability to cause infection.
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Molecular characterization of selected amoeba strains with emphasis to potential pathogens
KAŇKOVÁ, Hana
Goals of my work are: preparation of literature review, which will focus on free living amoebas as pathogens of humans and animals, molecular characterization of selected collection strains of amoebas: obtaining sequences of genes used as markers for these organisms (SSU rDNA, ITS). Data got by sequencing will be phylogenetically analyzed and results of analyzes interpreted. In the theoretical part I described the basic morphology of potentially pathogenic amoebas (acantamoebas, naegleriae), their pathogenicity and briefly also treatment of these diseases (primary amebic meningoencephalitis, granulomatous amebic encephalitis, acanthamoeba keratitis). Acanthamoebas and naeglerias (Naegleria fowleri and various species of acanthamoebas) are capable of entering human organism. Especially important is their ability to penetrate into brain, where they can be cause of amebic meningoencephalitis (disease of CNS). Primary amebic meningoencephalitis (PAM) is caused by N. fowleri. The diaseas occurs in children and young adults, who bathed in a permanently warmed water, where this species is found. Amoeba enter the body through the nasal cavity. PAM has rapid course human die within 7 days from first symptoms. Granulomatous amebic encephalitis (GAE) is caused by Acanthamoeba spp. The disease has a chronic character (its duration is up to several months). It is found in immunocompromised people (diabetes, HIV/AIDS, drug addiction ect.) Acanthamoebas enter through an injury and go to CNS by hematogenous route. Another disease caused by genus Acanthamoeba is acanthamoeba keratitis (AK). AK is inflammation of cornea. Acanthamoeba come in to the eye through contaminated contact lenses or when cornea is damaged. Treatment these diseases is very complicated and success rate is not high. Differences between acanthamoebas and naeglerias concern several areas life cycle, size and pathogenic potential. Naeglerias are able to create 3 stages (trophozoite, flagellate, cyst), acanthamoebas alternate between 2 stages (trophozoit, cyst). Trophozoite of acanthamoebas (12-35 ?m) is somewhat bigger in comparsion with naegleria (15-25 ?m). Major differences are visible on cysts which are irregular in shape in acanthamoebas and spherical and smooth in naeglerias. In the practical part of my work I try to answer several questions: determination of genotypes or species of amoebas that were isolated and to ascertain more information about their pathogenity, geographic range etc. In the methodical part I described procedures of cultivation of amoebas, isolation of their DNA, course of PCR, electrophoresis and software processing of sequences. In cultivation I used classic non-nutrient agar and amoebas were harvested after their visual checking.The PCR protocol was standard: I used specific primers and termocycler. PCR products were separated by electrophoresis. After cutting proper bands from the gel, I sent the sample to be sequenced by a specialized company (Macrogen). Final sequences were checked and sequences with high quality signal were processed by programs SeqMan, ClustalX, BioEdit. For phylogenetic analysis I used programs Paup, RAxML and MrBayes. I got 9 sequences of genes for SSU rRNA of acanthamoebas and 1 naegleria´s ITS sequence. Using phylogenetic analysis I got phylogenetic trees which I inserted to chapter with results. In the discussion chapter I outlined problems during gaining of naegleria´s sequences. The next part of discussion is devoted to detailed analysis of phylogenetic trees. Some of my sequences show close association with sequences derived from acanthamoebas which were isolated from patients with AK from various regions of the world, which indicate global distribution of some lineages of amoebas. Naegleria groups are more restricted by environment, but there are exceptions.
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