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Treatment and drug development against free-living pathogenic amoebae
Jankovcová, Klára-Marie ; Mach, Jan (advisor) ; Vinopalová, Martina (referee)
Free-living pathogenic amoebae are cosmopolitan unicellular eukaryotic organisms, which can cause several serious infections in humans and animals. They are causative agents of amoebic encephalitis, which are very rare and acute diseases of the central nervous system, that almost always lead to the death of the patient. Even though the importance of these pathogens has been increasing in recent years, the development of treatment procedures and drugs against them is very ineffective and unsatisfactory. Treatment and drug development is complicated mainly because of the complex biological structure of pathogens and limited knowledge of metabolic pathways and the biochemical reactions that are occurring in them, which are crucial for the development of effective drugs. In this bachelor thesis I focus on the pathogenic species Naegleria fowleri, Balamuthia mandrillaris and the pathogenic genus Acanthamoeba, which is the causative agent of encephalitis and acanthamoeba keratitis. In the first part of this bachelor thesis, I generally characterize the different representatives of free- living pathogenic amoebae and describe the clinical picture of individual diseases. In the second part I address the issue of treatment. Firstly, I describe current therapeutic options and issues of the treatment of...
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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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Molecular characterization of selected strains of amoebae of the genus Acanthamoeba, the potential human parasites.
ŠTAUBEROVÁ, Kamila
The genus Acanthamoeba belongs to a group of organisms generally called "amoebae". These amoebae can cause fatal disease in humans, being able to enter e.g. through skin in bloodstream and then spreading through the body. In the body they further parasitize in various tissues including the brain there they cause a little known disease called "granulomatous amoebic encephalitis". The disease is most prevalent in people previously ill with, e.g., systemic lupus erythematosus. Granulomatous amoebic encephalitis leads especially to a brain inflammation, which evolves in brain tissue necrosis. Most prominent symptoms are headaches, nausea and vomiting, aphasia and ataxia. This work mentions also some other amoebae as Neagleria fowleri, Balamuthia mandrillaris, Sappinia diploidea a Entamoeba histolytica. Neagleria fowleri is known as a causative agent of an illness called primary amoebic encephalitis. First, I had isolated DNA using Genomic Mini Kit from five amoeba strains. The next step was PCR amplification it is a method depending on alternation of high and lower temperatures in thermocycler. It was followed by electrophoresis, where the presence of bands in agarose gel, which I had prepared myself, showed the presence of absence of amoebic DNA fragments. In case of successful amplification (presence of PCR product) the fragment was excised from gel with scalpel and transferred in a test-tube. The DNA was then cleaned via extraction. The extraction was done with Gel/DNA Fragments Extraction Kit. An intermediate step before sequencing, we tried to clone the product it became obvious that this way is not very effective. The sequencing was provided by the company Seqme. Following work with sequences was conducted in programs BioEdit and alignment in ClustalX. Then, phylogenetic trees were computed in the program PAUP and viewed in TreeView. The results were interpreted in the terms of relationships between the amoebae majority of them belonged to the most common genotype, T4, but their relations within the genotype were not so tight. Concerning the clinical samples available to me (both from human eyes), one of them (O1) belonged to T4 genotype, whereas the other one (O2) belonged to the genotype T3. Another result was evaluation of the effectivity of different primers: specific primers AcaJDP1 and AcaJDP2 were much more efficient than originally used "eukaryotic" primers ERIB1 and ERIB10.
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