Made available in DSpace on 2014-06-11T19:22:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-04-18Bitstream added on 2014-06-13T18:08:05Z : No. of bitstreams: 1 garcia_jgd_me_arafcf.pdf: 502931 bytes, checksum: f5e5c5673828eed8be4251d55efd74a7 (MD5) / Universidade Estadual Paulista (UNESP) / O protozoário Giardia lamblia foi escolhido como tema de estudo, por ser um enteroparasita de prevalência significativa no mundo inteiro. Os objetivos desta pesquisa foram estudar a reprodutibilidade diagnóstica dos métodos Direto, Faust e cols., COPROTEST e Hematoxilina Férrica, bem como descrever a presença da Giardia lamblia, segundo a associação com algumas características da população de estudo como: grupo etário, gênero e distribuição dos casos segundo a variação sazonal nos meses em que se desenvolveu a pesquisa. Foram analisadas 200 amostras de fezes de crianças da região de Araraquara-SP e como resultados encontrou-se Giardia lamblia como o parasita mais freqüente, com 8,0%, não houve associação com o gênero; quanto à idade ocorreram mais casos no grupo de 03 a 05 anos e a maior freqüência de casos foi no mês de janeiro. Em relação as metodologias utilizadas chegou-se a conclusão que o melhor método diagnóstico para Giardia lamblia seria a associação de pelo menos, duas metodologias associadas de ótima reprodutibilidade que nesse estudo foram: COPROTEST - Faust; Direto-Faust e Coprotest-Direto (k > 0,81). / Giardia lamblia protozoan was chosen as a study them due to its significant prevalence all over the world. The present dissertation aims to study the diagnostic reproductibility of Direct, Faust et al., COPROTEST and Ferric Hematoxiline methods. It also describes the presence of Giardia lamblia, according to the association with some characteristics of the population in study, such as group age, gender and distribution of cases according to seasonal variation in the month studied. 200 feces samples of children from the region of Araraquara-SP were analysed and to cach of them the four diagnostic methodolgies were applied and then compared. From the results obtained it was possible to conclude that Giardia lamblia was the most frequent parasite, with 8, 0% occurrence; there was no association with gender; most cases of parasites were found in the 3-5 year-old groups, the highest frequency of parasites occurred in january and the best diagnostic to detect Giardia lamblia was the association of at least two methodologies of optimum reproductibility. The associations of optimum reproductibility were COPROTEST- Faust, Direct- Faust and COPROTEST- Direct (k >0, 81).

Resumo: O protozoário Giardia lamblia foi escolhido como tema de estudo, por ser um enteroparasita de prevalência significativa no mundo inteiro. Os objetivos desta pesquisa foram estudar a reprodutibilidade diagnóstica dos métodos Direto, Faust e cols., COPROTEST e Hematoxilina Férrica, bem como descrever a presença da Giardia lamblia, segundo a associação com algumas características da população de estudo como: grupo etário, gênero e distribuição dos casos segundo a variação sazonal nos meses em que se desenvolveu a pesquisa. Foram analisadas 200 amostras de fezes de crianças da região de Araraquara-SP e como resultados encontrou-se Giardia lamblia como o parasita mais freqüente, com 8,0%, não houve associação com o gênero; quanto à idade ocorreram mais casos no grupo de 03 a 05 anos e a maior freqüência de casos foi no mês de janeiro. Em relação as metodologias utilizadas chegou-se a conclusão que o melhor método diagnóstico para Giardia lamblia seria a associação de pelo menos, duas metodologias associadas de ótima reprodutibilidade que nesse estudo foram: COPROTEST - Faust; Direto-Faust e Coprotest-Direto (k > 0,81). / Abstract: Giardia lamblia protozoan was chosen as a study them due to its significant prevalence all over the world. The present dissertation aims to study the diagnostic reproductibility of Direct, Faust et al., COPROTEST and Ferric Hematoxiline methods. It also describes the presence of Giardia lamblia, according to the association with some characteristics of the population in study, such as group age, gender and distribution of cases according to seasonal variation in the month studied. 200 feces samples of children from the region of Araraquara-SP were analysed and to cach of them the four diagnostic methodolgies were applied and then compared. From the results obtained it was possible to conclude that Giardia lamblia was the most frequent parasite, with 8, 0% occurrence; there was no association with gender; most cases of parasites were found in the 3-5 year-old groups, the highest frequency of parasites occurred in january and the best diagnostic to detect Giardia lamblia was the association of at least two methodologies of optimum reproductibility. The associations of optimum reproductibility were COPROTEST- Faust, Direct- Faust and COPROTEST- Direct (k >0, 81). / Orientador: Maria Jacira Silva Simões / Coorientador: Vera Lucy de Santi Alvarenga / Banca: João Aristeu da Rosa / Banca: Ana Amélia Carraro Abrahão / Mestre

Giardia lamblia é um protozoário que acomete mais comumente animais jovens e que convivem em grupos. Apesar da alta prevalência, nem todos animais apresentam a doença clínica. Mesmo assim, a giardíase tem importância epidemiológica por possuir um elevado potencial zoonótico. O presente estudo teve como objetivo determinar a freqüência de Giardia lamblia em cães no município de Canoas, RS, Brasil, através do Método de Faust e cols. (1939) e da Técnica de Coloração da Auramina. Os grupos experimentais foram divididos de acordo com a procedência e o sexo. Das 332 amostras analisadas com o Método de Faust e cols, a estimativa em ponto da freqüência obtida foi de 34,04%, podendo variar de 28,95 a 39,13%, dentro de um intervalo de confiança de 95%. Destas amostras, 40,96% foram positivas em animais de canil e 27,11% de rua. O Teste Exato de Fisher aplicado a esses dados revelou existir uma diferença significativa (p = 0,0107) entre as variáveis resultado e procedência. A variável sexo, neste método não apresentou diferença significativa em relação ao resultado (p = 0,8162) totalizando 33,11% de machos positivos e 34,08% de fêmeas infectadas com o parasita. Das 147 amostras realizadas com a Técnica de Coloração da Auramina, 23 foram positivas, totalizando 15,65%. A análise estatística através do Teste McNemar revelou existir diferença significativa entre as duas técnicas (p = 0,0004). O valor Kappa foi igual a 0,07, considerado como um grau de concordância fraco. Os resultados encontrados neste estudo nos permitem afirmar que o Método de Faust e cols. foi o mais adequado para o diagnóstico na infecção por Giardia lamblia, entre os métodos analisados.

No description available.

[Abstract not supplied]

<p>The presence of human pathogens in water indicates the sanitary risk associated with different types of water utilization. This study surveyed the sources of human pathogens in urban waters. In order to evaluate the microbiological water quality of urban water, the enumeration of various indicator bacteria (total coliform, fecal coliform, E.coli and enterococci) is usually used.</p><p>The abundance of indicator bacteria in urban water indicates the level of fecal contamination and the presence of other human pathogens such as protozoan pathogens (Giardia lamblia & Cryptosporidium parvum).</p><p>Fecal pollution of urban waters can be from human and animal origin. Point sources of fecal contamination in an urbanized area are the effluents of urban wastewater treatment plants. While non-point sources are usually originated from diffuse sources such as (runoff from roads, parking lots, pets, leaks, failing septic systems and illegal sewer connections to storm drains). urban stormwater is considered as a major carrier for delivering human pathogens from diffuse sources to receiving waters. Increases in urban stormwater volumes have resulted from increasing urbanization and growth of impervious surfaces.</p><p>In order to reduce high amounts of human pathogens in urban waters, different methods are used nowadays to develop urban wastewater treatment plants technologies and urban stormwater management practices.</p>

The protozoan parasite Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the most common causes of diarrheal disease throughout the world, where an estimated 500 million people are infected annually. Despite efforts in trying to elucidate factors associated with virulence in G. intestinalis little is currently known. The disease outcome is highly variable in Giardia infected individuals, ranging from asymptomatic carriers to severe disease. The reasons behind the differences in disease outcome are vaguely understood and studies trying to link infectivity to different Giardia assemblages or sub-assemblages have rendered conflicting results. Prior to this study, little was known about the prevalence and genetic diversity of different G. intestinalis assemblages across the world. In this thesis, molecular characterization of clinical G. intestinalis samples from Eastern Africa and Central America, has been performed, enabling a better understanding of the prevalence of different Giardia genotypes in endemic areas (Papers I and II). A correlation between Giardia colonization and the presence of Helicobacter pylori in the human host was established. We found that the currently available genotyping tools provide low resolution when used to characterize assemblage A Giardia. Also, genotyping of assemblage B isolates at these loci is troublesome due to the polymorphic substitutions frequently found in the sequencing chromatograms. This ambiguity was investigated by using micromanipulation to isolate single assemblage B Giardia cells (Paper III). Both cultured trophozoites and cysts from giardiasis patients were analyzed. The data showed that allelic sequence heterozygosity (ASH) does occur at the single cell level, but also that multiple sub-assemblage infections appear to be common in human giardiasis patients. Furthermore, genome-wide sequencing followed by comparative genomics was performed in order to better characterize differences between and within different Giardia assemblages. The genome of a non-human infecting, assemblage E isolate (Paper IV) was sequenced.  The genomes of two freshly isolated human infecting assemblage AII isolates were also sequenced (Paper V). Subsequent, comparative analyses were performed and included the genomes of two human infecting isolates, WB (AI) and GS/M (B). Several important differences were found between assemblages A, B and E, but also within assemblage A; including unique gene repertoires for each isolate, observed differences in the variable gene families and an overall difference in ASH between the different isolates. Also, a new multi-locus genotyping (MLG) strategy for genotyping of assemblage A Giardia has been established and evaluated on clinical samples from human giardiasis patients.

The presence of human pathogens in water indicates the sanitary risk associated with different types of water utilization. This study surveyed the sources of human pathogens in urban waters. In order to evaluate the microbiological water quality of urban water, the enumeration of various indicator bacteria (total coliform, fecal coliform, E.coli and enterococci) is usually used. The abundance of indicator bacteria in urban water indicates the level of fecal contamination and the presence of other human pathogens such as protozoan pathogens (Giardia lamblia & Cryptosporidium parvum). Fecal pollution of urban waters can be from human and animal origin. Point sources of fecal contamination in an urbanized area are the effluents of urban wastewater treatment plants. While non-point sources are usually originated from diffuse sources such as (runoff from roads, parking lots, pets, leaks, failing septic systems and illegal sewer connections to storm drains). urban stormwater is considered as a major carrier for delivering human pathogens from diffuse sources to receiving waters. Increases in urban stormwater volumes have resulted from increasing urbanization and growth of impervious surfaces. In order to reduce high amounts of human pathogens in urban waters, different methods are used nowadays to develop urban wastewater treatment plants technologies and urban stormwater management practices.

This research aimed to address the knowledge gaps in the literature regarding the removal of waterborne pathogens (viruses and protozoa) by modified multistage slow sand filtration. In the current study, two pilot-scale multistage slow sand filtration systems were operated continuously for over two years. The pilot systems treated agricultural- and urban-impacted raw river water of variable quality with turbidity peaks over 300 NTU and seasonal cold temperatures <2°C. The first system (Pilot 1) consisted of two independent trains that included pre-ozonation, shallow-bed upflow gravel roughing filtration, and shallow-bed slow sand filtration. Pilot 1 was a pilot-scale version of an innovative, commercially available full-scale system. The second system (Pilot 2) included a full-depth upflow gravel roughing filter, a full-depth slow sand filter, and a second shallow-depth slow sand filter in series. The SSFs of both pilots were operated at high hydraulic loading rates (typically 0.4 m/h) at the upper limit of the literature recommended range (0.05 to 0.4 m/h). Both pilot systems provided excellent turbidity removal despite the high filtration rates. Effluent turbidity of all multistage SSF pilot systems were within the regulated effluent limits in Ontario for full-scale SSFs (below 1 NTU at least 95% of the time and never exceeded 3 NTU), despite raw water turbidity peaks over 100 NTU. The roughing filters contributed to approximately 60-80% of the full-train turbidity removal, compared to and 20-40% for the slow sand filters. On average, the second slow sand filter in pilot 2 provided almost no additional turbidity removal. The slow sand filter run lengths were short because of frequent high raw water turbidity, with about 50-80% of the runs in the range of 1-3 weeks. To prevent excessive SSF clogging and maintenance, filtration rates should be decreased during periods of high turbidity. Seven Cryptosporidium and Giardia challenge tests were conducted on the slow sand filters of both pilot systems at varying filtration rates (0.4 or 0.8 m/h), temperatures (2 to 25°C), and biological maturities (4 to 20 months). Removal of oocysts and cysts were good regardless of sand depth, hydraulic loading rate, and water temperature in the ranges tested. Average removals in the SSFs ranged from 2.6 to >4.4 logs for Cryptosporidium oocysts and ranged from >3.8 to >4.5 logs for Giardia cysts. This was consistent with findings in the literature, where oocyst and cyst removals of >4 logs have been reported. Cryptosporidium oocyst removals improved with increased biological maturity of the slow sand filters. At a water temperature of 2°C, average removal of oocysts and cysts were 3.9 and >4.5 logs, respectively, in a biologically mature SSF. Doubling the filtration rate from 0.4 to 0.8 m/h led to a marginal decrease in oocyst removals. Sand depths in the range tested (37-100 cm) had no major impact on oocyst and cyst removals, likely because they are removed primarily in the upper section of slow sand filter beds by straining. In general, good oocyst and cyst removals can be achieved using shallower slow sand filter bed depths and higher filtration rates than recommended in the literature. There are very few studies in the literature that quantify virus removal by slow sand filtration, especially at high filtration rates and shallow bed depths. There are no studies that report virus removal by slow sand filtration below 10°C. As such, 16 MS2 bacteriophage challenge tests were conducted at varying water temperatures (<2 to >20°C) and filtration rates (0.1 vs. 0.4 m/h) between February and June 2006 on biologically mature slow sand filters with varying bed depths (40 vs. 90 cm). Biologically mature roughing filters were also seeded with MS2. Average MS2 removals ranged from 0.2 to 2.2 logs in the SSFs and 0.1 to 0.2 logs in the RFs under all conditions tested. Virus removal by slow sand filtration was strongly dependant on hydraulic loading rate, sand depth, and water temperature. Virus removal was greater at a sand depth of 90 cm vs. 40 cm, at an HLR of 0.1 m/h vs. 0.4 m/h, and at warm (20-24°C) vs. cold (<2-10°C) water temperatures when sufficient warm water acclimation time was provided. Increased sand depth likely increased MS2 removal because of greater detention time for predation and greater contact opportunities for attachment to sand grains and biofilms. A lower HLR would also increase MS2 removal by increasing detention time, in addition to decreasing shear and promoting attachment to filter media and biofilms. Greater MS2 removal at warmer water temperatures was attributed to improved biological activity in the filters. Schmutzdecke scraping was found to have only a minor and short-term effect on MS2 removals. Virus removal can be optimized by providing deep SSF beds and operating at low filtration rates. Virus removal may be impaired in cold water, which could affect the viability of using SSF/MSF at northern climates if communities do not use disinfection or oxidation. As a stand-alone process, slow sand filtration (with or without roughing filtration) may not provide complete virus removal and should be combined with other treatment processes such as disinfection and oxidation to protect human health.

The diplomonads are a diverse group of eukaryotic flagellates found in microaerophilic and anaerobic environments. The most studied diplomonad is the intestinal parasite Giardia intestinalis, which infects a variety of mammals and cause diarrheal disease. Less is known about Spironucleus salmonicida, a parasite of salmonid fish, known to cause systemic infections with high mortality. We created a transfection system for S. salmonicida to study cellular functions and virulence in detail (Paper I). The system was applied to explore the mitochondrion-related organelle (MRO) in S. salmonicida. We showed that S. salmonicida possesses a hydrogenosome (Paper II) with a higher metabolic capacity than the corresponding MRO of Giardia, the mitosome. Evolutionary analysis of key hydrogenosomal proteins showed ancient origin, indicating their presence in the ancestral diplomonad and subsequent loss in Giardia. Annexins are of evolutionary interest since these proteins are found across all kingdoms. Annexin-like proteins are intriguingly expanded into multigene families in Giardia and Spironucleus. The annexins of S. salmonicida were characterized (Paper III) with distinct localizations to various cellular structures, including a putative adhesion structure anterior in the cell. The disease-causing Giardia trophozoites differentiate into infectious cysts, a process essential for transmission and virulence of the parasite. Cysts are often spread via contaminated water and exposed to environmental stressors, such as UV irradiation. We studied the survival and transcriptional response to this stress factor (Paper IV) and results showed the importance of active DNA replication machinery for parasite survival after DNA damage. In addition, we studied transcriptional changes along the trajectory of encystation (Paper V), which revealed a coordinated cascade of gene regulation. This was observed for the entire transcriptome as well as putative regulators. Large transcriptional changes appeared late in the process with the majority of differentially regulated genes encoding hypothetical proteins. We studied the localizations of several of these to gain information of their possible function. To conclude, the diplomonads are complex eukaryotic microbes with cellular processes adjusted to match their life styles. The work in this thesis has provided insight of their adaptations, differences and similarities, but also new interesting leads for future studies of diplomonad biology and virulence.