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The development of molecular tools for detection and typing of unicellular intestinal parasites has revealed genetic diversities in species that were previously considered as distinct entities. Of great importance is the genetic distinction found between the pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar, two morphologically indistinguishable species. Blastocystis sp. is a ubiquitous intestinal parasite with unsettled pathogenicity. Molecular studies of Blastocystis sp. have identified 17 genetic subtypes, named ST1-17. Genetically, these subtypes could be considered as different species, but it is largely unknown what phenotypic or pathogenic differences exist between them. This thesis explores molecular methods for detection and genetic subtyping of unicellular intestinal parasites, with special focus on Blastocystis. We found that PCR-based methods were highly sensitive for detection of unicellular intestinal parasites, but could be partially or completely inhibited by substances present in faeces. A sample transport medium containing guanidinium thiocyanate was shown to limit the occurrence of PCR inhibition. The prevalence of Blastocystis in Swedish university students was over 40%, which is markedly higher than what was previously estimated. Blastocystis ST3 and ST4 were the two most commonly found Blastocystis subtypes in Sweden, which is similar to results from other European countries. Blastocystis sp. and Giardia intestinalis were both commonly detected in Zanzibar, Tanzania, each with a prevalence exceeding 50%. Blastocystis ST1, ST2, and ST3 were common, but ST4 was absent. While G. intestinalis was most common in the ages 2-5 years, the prevalence of Blastocystis increased with increasing age, at least up to young adulthood. We found no statistical association between diarrhoea and Blastocystis sp., specific Blastocystis subtype or G. intestinalis. Metagenomic sequencing of faecal samples from Swedes revealed that Blastocystis was associated with high intestinal bacterial genus richness, possibly signifying gastrointestinal health. Blastocystis was also positively associated with the bacterial genera Sporolactobacillus and Candidatus Carsonella, and negatively associated with the genus Bacteroides. Blastocystis ST4 was shown to have limited intra-subtype genetic diversity and limited geographic spread. ST4 was also found to be the major driver behind the positive association between Blastocystis and bacterial genus richness and the negative association with Bacteroides.

Orientador: Semíramis Guimarães Ferraz Viana / Resumo: Giardia duodenalis é um dos principais agentes etiológicos de diarreia infecciosa, sobretudo em crianças em idade pré-escolar que vivem em comunidades de baixa renda. Estudos da diversidade genética de G. duodenalis ampliaram o conhecimento da epidemiologia nas infecções humanas, entretanto um dos temas mais interessantes e menos conhecidos é a possível interação de Giardia com o microbioma do hospedeiro e com patógenos concomitantes. No presente estudo, avaliou-se a composição e a diversidade da comunidade bacteriana de crianças saudáveis e crianças com diarreia, parasitadas por Giardia e outros protozoários intestinais. Os isolados de Giardia obtidos nessa população foram caracterizados geneticamente. Amostras de fezes foram obtidas de 181 crianças de 0 a 6 anos de idade, das quais 156 crianças hígidas atendidas em centros de educação infantil e 25 crianças com diarreia atendidas no PS Infantil Municipal. Cada amostra de fezes foi processada para o exame microscópico e submetida à extração de DNA a ser empregado em duas etapas distintas: (1) amplificação e sequenciamento Sanger para a caracterização genética de Giardia e o diagnóstico de Blastocystis sp, Dientamoeba fragilis, Enterocytozoon bieneusi e Cryptosporidium spp. e (2) amplificação do gene 16s RNA ribossomal e sequenciamento de nova geração (plataforma Illumina MiSeq) para a caracterização da microbiota intestinal. Giardia (36,5%) e Blastocystis (41,7%) foram os parasitas mais prevalentes. A caracterização genética... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Giardia duodenalis is one of the major etiological agents of infectious diarrhea, especially in preschool children living in low-income settings. Studies focused on genetic diversity of G. duodenalis have provided insights for a better understanding of epidemiology in human infections. However, one of the most interesting and least known issues is the possible interplay between Giardia and the host microbiome and concomitant pathogens. In this work, we evaluated the diversity and composition of bacterial community of healthy children and children presenting with diarrhea infected by Giardia and/or other intestinal protozoa. In addition, Giardia isolates infecting this population were genotyping. A total of 181 stool samples from children aged 0 to 6 years old (156 from daycare children and 25 from diarrheic children attending in an emergence pediatric center) were tested by microscopic examination and submitted to DNA extraction for the following steps: (1) conventional PCR/sequencing for Giardia genotyping and the diagnosis of Blastocystis sp, Dientamoeba fragilis, Enterocytozoon bieneusi and Cryptosporidium spp and (2) next-generation sequencing (Illumina MiSeq) based analysis of intestinal microbiota. Giardia (36.5%) and Blastocystis (41.7%) were the most prevalent parasites. Analysis of Giardia sequences retrieved from 61 isolates revealed infections by assemblages A (31%), B (69%) and mixed infections A+B (3%). Metagenomic analyzes revealed similarity of bacterial microb... (Complete abstract click electronic access below) / Doutor

The diplomonads are a diverse group of eukaryotic microbes found in oxygen limited environments such as the intestine of animals were they may cause severe disease. Among them, the prominent human parasite Giardia intestinalis non-invasively colonizes the small intestine of humans and animals where it induces the gastrointestinal disease giardiasis. Two of the eight genetic groups of G. intestinalis, assemblage A and B, are known to infect humans and have zoonotic potential. At the start of project, genome scale data from assemblage B-H was either sparse or entirely missing. In this thesis, genome sequencing was performed on the assemblage B isolate GS (Paper I) and the P15 isolate (Paper III) of the hoofed-animals specific assemblage E to investigate the underlying components of phenotypic diversity in Giardia. Comparisons to assemblage A isolate WB revealed large genomic differences; entirely different repertoires of surface antigens, genome rearrangements and isolate specific coding sequences of potential bacterial origin. We established that genomic differences are also manifested at the transcriptome level (Paper VIII). In a follow up analysis (Paper IV) we concluded that the Giardia assemblages are largely reproductively isolated. The large genomic differences observed between Giardia isolates can explain the phenotypic diversity of giardiasis. The adaptation of diplomonads was further studied in Spironucleus barkhanus (Paper II), a fish commensal of grayling, that is closely related to the fish pathogen Spironucleus salmonicida, causative agent of systemic spironucleosis in salmonid fish. We identified substantial genomic differences in the form of divergent genome size, primary sequence divergence and evidence of allelic sequence heterozygosity, a feature not seen in S. salmonicida. We devised a transfection system for S. salmonicida (Paper VI) and applied it to the study of the mitochondrial remnant organelle (Paper VII). Our analyses showed that S. salmonicida harbor a hydrogenosome, an organelle with more metabolic capabilities than the mitosome of Giardia. Phylogenetic reconstructions of key hydrogenosomal enzymes showed an ancient origin, indicating a common origin to the hydrogenosome in parabasilids and diplomonads. In conclusion, the thesis has provided important insights into the adaptation of diplomonads in the present and the distant past, revealing hidden diversity.

Orientadores: Regina Maura Bueno Franco, Romeu Cantusio Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T01:13:52Z (GMT). No. of bitstreams: 1 Yamashiro_Sandra_M.pdf: 8805357 bytes, checksum: 269acfd8ffaefb317f8e7f96a90785f7 (MD5) Previous issue date: 2010 / Resumo: O reservatório de Salto Grande é formado pelo rio Atibaia que contribui para a poluição das águas superficiais desta represa, devido ao despejo de efluentes domésticos. Além disso, o desenvolvimento urbano, a industrialização e atividade agrícola nas proximidades da represa, também proporcionam alteração da qualidade das águas deste manancial. Os protozoários gastrointestinais, Giardia spp. e Cryptosporidium spp. estão amplamente presentes em ambiente aquático e suas formas infectantes, cistos e oocistos, são resistentes às condições ambientais (temperatura, radiação solar) e ao processo de desinfecção da água (principalmente à cloração). Inúmeros surtos de gastroenterite foram causados por estes organismos devido à veiculação hídrica sendo que estes protozoários patogênicos tornaram-se uma preocupação constante para os sistemas produtores de água. Os objetivos desta pesquisa foram: verificar a ocorrência de Giardia spp. e Cryptosporidium spp. nas águas superficiais em quatro locais da represa de Salto Grande (Ponto 1: Mini-Pantanal, Ponto 2: Saltinho, Ponto 3: Praia Azul e Ponto 4: Iate Clube) com periodicidade mensal (12 meses); avaliar a qualidade da água mediante análise microbiológica e físico-química deste manancial; comparar a ocorrência destes protozoários nos diferentes pontos (1, 2, 3 e 4); verificar a correlação entre Giardia, Cryptosporidium e os parâmetros analisados; verificar a aplicabilidade do método de filtração em membrana para detecção dos protozoários em um manancial eutrofizado; comparar qualitativamente dois kits comerciais de anticorpos monoclonais para detecção destes agentes parasitários. Para os ensaios parasitológicos, foi empregada a técnica de filtração em membrana de acordo com Franco et al (2001) e visualização mediante reação de imunofluorescência direta com anticorpos monoclonais comerciais; 2) para os ensaios microbiológicos visando a detecção de coliformes totais, termotolerantes e/ou Escherichia coli utilizando as técnicas de tubos múltiplos de acordo com os procedimentos descritos no "Standard Methods" (APHA,AWWA,WEF 2005) e; 3) a contagem de células de cianobactérias foi feita por sedimentação em câmera de Utermöhl, e utilização de microscópio invertido e retículo de Whipple (CETESB, Normalização técnica-NT-06.L5.303,2005). A análise estatística foi realizada com o emprego das técnicas de Análise de Variância, Teste de Comparação Múltipla de Médias (Teste de Duncan), Teste de Qui-Quadrado, Correlação de Pearson com o objetivo de comparar a positividade para os protozoários nos diferentes pontos de coleta e verificar a existência de correlação entre parâmetros biológicos, físico-químicos e parasitológicos das amostras de água do reservatório. De janeiro a dezembro de 2008, 48 amostras de água foram colhidas. Nos pontos 1 e 2, cistos de Giardia foram detectados em 16,6% (326 cistos/L) e 41,6% (785 cysts/L) das amostras, respectivamente. Oocistos de Cryptosporidium foram detectados somente no ponto 2, em 8,3% das amostras (170 cistos/L) enquanto nos pontos 3 e 4 não foram encontrados. A técnica de filtração em membranas apresentou eficiência de recuperação de 8,2% a 34,1% para cistos de Giardia e 5,3% a 8,3% para oocistos de Cryptosporidium (pontos 1 e 2) e para os pontos 3 e 4, as recuperações foram de 42,6% a 88,6% para cistos e 31,9% a 87,5% para oocistos. A separação imunomagnética não resultou em recuperação dos protozoários nas amostras de água que apresentaram floração de cianobactérias (pontos 3 e 4). Nas amostras de água dos pontos 1, 3 e 4, não foram encontradas correlações entre os protozoários e coliformes enquanto no ponto 2, houve correlação entre cistos de Giardia, coliformes termotolerantes e E.coli. / Abstract: The Salto Grande Reservoir is formed by the Atibaia River, that contributes to the pollution of this superficial water reservoir due to the discharge of domestic effluents. In addition, urban development, industrialization, and agricultural activity in the vicinity of the dam also cause degradation in water quality of this watershed. The gastrointestinal pathogenic protozoa Giardia spp. and Cryptosporidium spp are ubiquitous in aquatic environments and their infectious forms, cysts and oocysts, are resistant against environmental conditions (temperature, solar radiation) and water disinfection process (chlorination). Numerous outbreaks of gastroenteritis have been caused by these organisms due to waterborne transmission, and these parasitic protozoa have become a permanent concern in water treatment plants The aims of this study were: to investigate the occurrence of Giardia cysts and Cryptosporidium oocysts in superficial water samples of 4 sites of Salto Grande Reservoir (Point 1: Mini-Pantanal, Point 2: Saltinho, Point 3: Praia Azul and Point 4: Iate Clube) in 12 month period; to assess water quality by microbiological and physical-chemical analysis of water samples from 4 points of this reservoir; to compare the occurrence of these protozoa in different points (1, 2, 3 and 4); to verify the correlation among Giardia, Cryptosporidium and the analyzed parameters; to examine the applicability of the membrane filtration technique for the detection of protozoa in an eutrophic watershed; to qualitatively compare two commercial kits of monoclonal antibodies for detecting these parasites. For parasitological assays, the membrane filter technique according to Franco et al (2001) was employed following visualization by direct immunofluorescence assay (IFA) with commercial monoclonal antibody for enumerating cysts and oocysts in various samples; 2) for microbiological tests aiming the detection of total coliforms, thermotolerant and/or E.coli, the techniques of multiple tubes were used according to the procedures described in "Standard Methods" (APHA, AWWA, WEF, 2005) and 3) cyanobacteria cells counting was performed with Utërmohl Chamber and inverted microscopy and Whipple Grid (CETESB, Normalização técnica-NT-06.L5.303, 2005). Statistical analysis was performed using Analysis of Variance, Multiple Comparisons Test (Duncan's test), Chisquare Test and Pearson Correlation, in order to compare the occurrence of protozoa in different points, and to verify the presence of correlation among biological parameters, physical-chemical, and the presence of parasitic protozoa in water samples from the reservoir. From January to December in 2008, 48 water samples were collected. In points 1 and 2 (Mini-Pantanal e Saltinho), Giardia cysts were detected in 16,6% (326 cistos/L) and 41,6% (785 cysts/L) of the samples, respectively. Cryptosporidium oocysts were detected only in point 2 (Saltinho) in 8,3% (170 oocysts/L) of the samples, while in the others points (3 e 4), both parasites were not detected. The membrane filtration technique attained recovery efficiency range of 8,22% to 34,18% for Giardia cysts and 5,3% to 8,3% Cryptosporidium oocysts (points 1 and 2) and for points 3 and 4, the recoveries efficiency were 42,63% to 88,66% for cysts and 31,97% to 87,5% for oocysts. The immunomagnetic separation did not result in recovering of protozoa in water samples with cyanobacteria blooms (points 3 and 4). In water samples from points 1, 3, and 4, no correlations were found between parasites concentration and coliforms. Water samples from point 2, in contrast, had correlation among Giardia cysts, thermotolerant coliforms, and E.coli. / Mestrado / Parasitologia / Mestre em Parasitologia

Made available in DSpace on 2014-07-29T15:13:43Z (GMT). No. of bitstreams: 1 Dissertacao Vanessa Silvestre Ferreira de Oliveira.pdf: 1341350 bytes, checksum: 9ce3345b7f150b232b983b427448e976 (MD5) Previous issue date: 2011-03-16 / Este trabalho teve como objetivos identificar os protozoários encontrados em fezes de cães, observar a ocorrência da infecção por Neospora caninum e Toxoplasma gondii em cães pela Reação de Imunofluorescência Indireta (RIFI), desenvolver e validar técnicas de ELISA para o diagnóstico sorológico de N. caninum em cães, determinar a utilidade do Western Blot para esse diagnóstico e observar a ocorrência de DNA de N. caninum, T. gondii e Hammondia heydorni em tecido cerebral de cães. Para isso, foram colhidas amostras de cérebro e sangue de 251 cães, e fezes de 106 cães, provenientes do Centro de Zoonoses de Goiânia, Goiás. Foram utilizados oito soros de infecção experimental por N. caninum e quatro por T. gondii para comprovação dos resultados sorológicos. As amostras de fezes foram concentradas pelo método de Teleman seguido da flutuação com solução de Sheather, para visualização dos oocistos. Foi realizada a extração de DNA de todas as amostras, posteriormente analisadas por nested-PCR específicas para detecção de DNA de N. caninum, H. heydorni, T. gondii, Cryptosporidium spp. e Giardia spp. Uma nested-PCR foi ainda realizada nas amostras em que foram visualizados oocistos menores de 20 µm, para a detecção de DNA de coccídios e posterior seqüenciamento, para correta identificação dos mesmos. A técnica de RIFI foi realizada em todas as amostras de sangue, e técnicas de ELISA foram padronizadas, utilizando-se extrato solúvel de taquizoítos e as proteínas recombinantes rNcGRA7, rNcSAG4, rNcBSR4 e rNcSRS9 de N. caninum. Para a detecção de DNA dos parasitos nos cérebros, estes foram divididos em três partes, e de cada parte foi realizada a extração de DNA. Após a extração, foram realizadas nested-PCR específicas para cada parasito. Nas fezes dos cães, foram encontradas doze amostras contendo oocistos. Em seis, os oocistos eram maiores que 20 µm, e foram identificados como de Cystoisospora canis, e nas outras seis, oocistos menores que 20 µm foram visualizados e identificados como sendo quatro de H. heydorni, um de Cystoisospora spp. e uma amostra apresentava DNA tanto de H. heydorni quanto de Cystoisospora spp. DNA de C. canis foi identificado em quatro amostras, uma amostra foi identificada como G. duodenalis genótipo C e quatro como G. duodenalis genótipo D. Nenhuma amostra de fezes foi positiva para N. caninum e T. gondii, e quatro foram positivas para H. heydorni na PCR específica para este parasito. Nos cérebros analisados, foram encontradas seis amostras (2,4%) positivas a N. caninum e 23 amostras (9,1%) foram positivas a T. gondii. Nenhuma amostra foi positiva a H. heydorni. Anticorpos anti-N. caninum foram encontrados em 18,32% dos cães analisados, e 56,57% dos cães apresentaram sorologia positiva a T. gondii pela técnica de RIFI. Técnicas de ELISA foram padronizadas, utilizando-se extrato solúvel de taquizoítos e as proteínas recombinantes rNcGRA7, rNcSAG4, rNcBSR4 e rNcSRS9 de N. caninum. Foram encontrados valores de sensibilidade e especificidade menores que 84%, além do reconhecimento das proteínas recombinantes pelos dois parasitos, nos soros de infecções naturais e experimentais. Uma baixa concordância entre as técnicas foi observada, com valores de Kappa inferiores a 0,4. Os resultados encontrados indicam que N. caninum está menos presente em fezes de cães, enquanto H. heydorni é mais facilmente encontrada. As amostras que continham Cryptosporidium e Giardia foram identificadas como espécies-específicas. A pesquisa de DNA de parasitos em órgãos de cães pode ser utilizada como uma ferramenta para o diagnóstico post-mortem de infecções por protozoários, porém as técnicas de PCR podem apresentar baixa sensibilidade devido à pequena quantidade de tecido utilizado. Os resultados encontrados no WB indicam não haver um padrão de reconhecimento das proteínas imunodominantes, e que as técnicas sorológicas na neosporose canina têm limitações, devido aos baixos títulos encontrados e à reação cruzada com T. gondii, além da alta prevalência da infecção por T. gondii em cães, e presença de infecções mistas.

Underdiagnosis of intestinal parasites in patients with diarrhea A compilation from the Swedish public health authority indicates that infections caused by Cryptosporidium spp. increased in Sweden from 47 cases in 2004 to 594 cases in 2016 and Giardia intestinalis causes around 1300 infections per year. The primary aim of this study was to investigate the prevalence of parasites in patients with diarrhea. Furthermore, the study investigated whether samples taken with E-swab could be analyzed with real-time polymerase chain reaction (PCR) for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica/dispar and G. intestinalis rather than Sodium acetate-acetic acid-formaline fixative (SAF-fixative). Prevalence of parasites in fecal samples was collected from 200 samples from patients with bacterial issue ordered. For evaluation of E-swab, 22 frozen, unfixed samples that were positive for intestinal parasites was used. Twelve positive E-swab samples was used as comparative positive controls. This was analyzed using real-time PCR. Bacteria was counted for 9.5% of the infections whilst parasites counted for 14% of the infections. The conclusion was that E-swab could replace SAF-fixative in the diagnosis of intestinal parasites and that there is that an underdiagnosis of intestinal parasites. Keywords: Cryptosporidium spp, Dientamoeba fragilis, Entamoeba histolytica, Entamoeba dispar, Giardia intestinalis, real-time PCR, E-swab, prevalence.

Les analyses de risque microbiologique, dont l'ÉQRM (évaluation quantitative du risque microbien) proposent de nouvelles techniques pour évaluer les conséquences sanitaires liées à la contamination microbiologique de l'eau potable. Ces modèles intègrent les données physico-chimiques et microbiologiques des usines de traitement d'eau pour quantifier un risque à la santé. Le projet visait à évaluer le lien entre le risque estimé selon un modèle ÉQRM et l’incidence de giardiase observée. Les banques de données des maladies à déclaration obligatoire et d’INFO-SANTÉ ont été utilisées pour comparer le résultat de l’analyse de risque à celui des analyses épidémiologiques. Les municipalités considérées les plus à risque par l'ÉQRM ont une incidence de gastroentérite et de parasitoses plus élevée. Cependant, l'ampleur du risque prédit ne correspond pas à celui observé. Il est souhaitable que les modèles d’ÉQRM incorporent des données populationnelles pour prédire avec une plus grande exactitude le risque épidémiologique.

Die Durchfallerkrankung Giardiasis wird durch den Protisten Giardia duodenalis ausgelöst. Die Infektion erfolgt fäkal-oral, meist über kontaminiertes Trinkwasser. Der Parasit kolonisiert den oberen Bereich des Dünndarms und heftt sich an das Epithel, wodurch es die Krankheitsbeschwerden auslöst. Allerdings sind Details über die Mechanismen der Pathogenese unbekannt. Dazu kommt, dass der Ausgang einer Infektion fallspezifisch starken Schwankungen unterworfen ist, von selbst-limitierend bis chronisch und asymptomatischer Kolonisierung bis hin zur schweren Enteritis. Ein möglicher Pathomechanismus ist der Wegfall der Barrierefunktion des Dünndarmepithels, z.B. durch Beeinträchtigung von tight junctions oder Zelltod. In dieser Arbeit wurden Effekte von G. duodenalis auf in vitro Modellsysteme des humanen Dünndarmepithels untersucht. Dazu wurden hauptsächlich Daten über die Barrierefunktion sowohl von der weit verbreiteten Caco-2 Zelllinie, als auch über ein neu etabliertes humanes Dünndarmorganoidsystem, erhoben. Es konnte gezeigt werden, dass mehrere - mitunter in der Literatur als hochvirulent beschriebene - G. duodenalis Isolate zu keinerlei Beeinträchtigung der Barrierefunktion oder irgendeiner anderen untersuchten potenziellen Schädigung an zwei unterschiedlichen Caco-2 Zelllinien unter diversen Infektions- und Kulturbedingungen führte. Jedoch andererseits das neu entwickelte Dünndarmorganoidsystem mit pseudo-luminalem Medium TYI S 33 reproduzierbar die Zerstörung des Epithelmodells mit Zellverlust, Zelltod (apoptotisch und nicht-apoptotisch), Störung der tight junctions (Abbau und Dislokation von Claudinen und ZO-1) und den Verlust von Mikrovilli innerhalb ein bis zwei Tage nach Parasiteninfektion zeigen konnte. Zudem wurde das Auftauchen von ClCa-1-Signalen unter andauerndem Infektionsstress beobachtet, was die Differenzierung bzw. Metaplasie zu Becherzellen nahelegt, jedoch keine Wirtsreaktion auf die Gewebszerstörung zu sein scheint. / The protozoan parasite Giardia duodenalis is the etiological agent for the intestinal diarrheal disease giardiasis. Infections are acquired via the fecal-oral route, mostly via uptake of cysts from contaminated drinking water. The colonization of the hosts’ duodenum and upper jejunum and the attachment of Giardia trophozoites onto the epithelium is the cause of a variety of gastrointestinal complaints but the exact pathomechanisms are unknown. Furthermore, the outcome of Giardia infections varies greatly between individuals, ranging from self-limiting to chronic, and asymptomatic to severe enteritis. One proposed mechanism for the pathogenesis is the breakdown of intestinal barrier function, e.g. by tight junction impairment or induction of cell death. In this work, effects of G. duodenalis on in vitro models of the human small intestinal epithelium were investigated by studying mainly barrier-related properties and changes of widely used Caco-2 cells as well as newly established human small intestinal organoid-derived monolayers (ODMs). It could be shown that several isolates of G. duodenalis, some described as highly virulent, fail to induce barrier dysfunction or any other investigated pathological effect on two Caco-2 cell lines under various infection and culturing conditions. On the other side, by developing a new organoid-based model system and the use of luminal mock medium TYI-S-33, considerable epithelial disruption (including loss of cells), cell death (apoptosis and non-apoptotic), tight junction impairment (degradation and dislocation of claudins and ZO-1), and microvilli depletion reproducibly induced by G. duodenalis trophozoites between one and two days after infection could be observed. Moreover, emergence of ClCa-1 positive cells with ongoing parasite infections suggest epithelial differentiation or metaplasia towards goblet cells, which is furthermore not associated to tissue damage.

Thesis. (M. Tech. (Dept. of Biosciences, Faculty of Applied and Computer Sciences))--Vaal University of Technology, 2010 / Methods for detecting the presence of Cryptosporidium oocysts and Giardia cysts have been developed and continuous improvement is being done to improve the recovery rate of the target protozoa. Rand Water has adopted their method for isolation and detection of Cryptosporidium oocysts and Giardia cysts in water from United State Environmental Protection Agency (US-EPA) Method 1623, 1999. In 2005 changes were made by US-EPA to the Method 1623. A study was done to improve the performance of the Rand Water Method 06 (2007) used for isolation and detection of Cryptosporidium oocysts and Giardia cysts. Three methods namely: Rand Water Method 06 (2007), US-EPA Method 1623 (2005) and Drinking Water Inspectorate standard operating procedures (2003) were compared and key different steps in the methods were identified (wrist action speed, centrifuge speed, immunomagnetic separation procedures and addition of pre-treatment steps). Different experiments were conducted to verify and evaluate the difference between two wrist action shaker speeds, three different centrifuge speeds, two slightly different immunomagnetic separation procedures and when a pre-treatment step was included in the method. Three different types of water matrices (reagent grade water, drinking water and raw water) were used for the experiments and secondary validation. Data obtained from the experiments and secondary validation was statistically analyzed to determine whether there was a significant difference in the recovery of Cryptosporidium oocysts and Giardia cysts. Secondary validation of the Rand Water Method 06 (2007) was performed by implementing the study experiments‟ findings into the method. The results indicated an increase in the recovery rate of Cryptosporidium oocysts and Giardia cysts when data was compared with the previous secondary validation report. The mean recovery of Cryptosporidium oocysts in reagent grade water samples increased from 31% to 55%, drinking water samples increased from 28% to 44% and raw water decreased from 42% to 29%. The mean recovery of Giardia cysts in reagent grade water samples increased from 31% to 41%, drinking water samples increased from 28% to 46% and raw water decreased from 42% to 32%. Furthermore, even though the recovery rate of raw water decreased the use of pre-treatment buffer reduced the number of IMS performed per sample by reducing the pellet size. Enumeration of microscope slides was also easier as there was less background interference. The optimization of the Rand Water Method 06 (2007) was successful as the recovery rate of Cryptosporidium oocysts and Giardia cysts from water increased. All the changes that were verified and that increased the recovery rate were incorporated into the improved Rand Water Method 06.