Giardia lamblia, a worldwide cause of diarrhoea, must differentiate into environmentally resistant cysts for dissemination and completion of its life cycle. Although G. lamblia is an early diverging eukaryote, encystation involves many complex cellular changes including formation of the cyst wall that contains at least two cyst wall proteins, cyst wall proteins 1 and 2. Cwp genes are transcribed only during encystation. In this study, we examine the regulatory elements for the encystation-specific gene cwp2. The 64 bp immediately upstream of the cwp2 open reading frame (-64 to -1 relative to ATG) was shown to be sufficient for the encystation-specific expression of luciferase. To determine which region(s) within this 64 bp contributed to encystation-specific expression in vivo, a series of deletions were cloned into a Giardia luciferase expression vector and their ability to control encystation-specific expression of luciferase was assessed. Deletion of elements in the -64 to -23 region of the cwp2 promoter significantly increased expression of luciferase in vegetative trophozoites, suggesting that this area contains a negative cis-acting element. Deletions of elements from -23 to -10 led to decreased expression in encysting cells, suggesting that this region may contain positive cis-acting elements. When the A/T-rich initiator was deleted but the cis-acting elements (-64 to -10) were retained, encystation-specific expression of luciferase was maintained but an aberrant transcriptional start site was utilised. These results indicate that Giardia has developed a classic repressor mechanism(s) that allows tight, encystation-specific control by the cwp2 promoter.

Zur Klärung der Frage, welche Rolle die Annexinen-homologen alpha-Giardine 14 und 19 tatsächlich im Lebenszyklus des humanpathogenen Darmparasits Giardia lamblia einnehmen, wurden verschiedene molekularbiologische und biochemische Untersuchungen durchgeführt. Die Untersuchungen im Falle des alpha14-Giardins ergaben, dass das Protein nur in gewissen Regionen der membranumgebenen Bereiche der Flagellen als Perlenschnur-ähnliche Verdickungen vorlag. Zusätzlich konnte sowohl eine Assoziation mit den Mikrotubuli des Axonems als auch mit der Plasmamembran nachgewiesen werden. Bei der Fahndung nach direkten Interaktionspartnern des alpha14 belegten verschiedene Methoden eine Wechselwirkung zwischen der Ankyrin-Domäne einer Ser/Thr-Kinase und dem alpha14. Auch konnte eine Phosphorylierung des Giardins eindeutig bestätigt werden. Weitere Eigenschaften des alpha14 waren die Fähigkeit der Oligomerisierung als auch die Bindung an Glykosaminoglykane. Infolgedessen könnte das alpha14-Giardin eine durch Phosphorylierung regulierte MAP-Funktion übernehmen und somit eine wichtige Rolle für die Dynamik oder Mobilität der Flagellen spielen. Das Hauptziel der im Falle des alpha19-Giardin durchgeführten Untersuchungen war die erste biochemische Charakterisierung des Proteins. Nach Bestätigung der Expression des alpha19-Giardins konnte mit Hilfe eines spezifischen Antikörpers das Protein ausschließlich in den ventralen Flagellen von G. lamblia nachgewiesen werden. Im Weiteren wurde für das alpha19 der typischen Annexin-Charakter sowie eine Phosphorylierung bestätigt. Das Vorkommen des alpha19-Giardins in der Membranfraktion des G. lamblia-Rohextraktes sowie die Solubilisierung des Proteins durch Zugabe eines Detergenz gaben erste Hinweise auf eine Fettsäuremodifikation des N-Terminus des Proteins, in dem ein Sequenzmotiv für eine Myristoylierung vorliegt. Folglich könnte das Protein an membrandynamischen Prozessen innerhalb der ventralen Flagellen beteiligt sein.

Der Intestinalparasit Entamoeba histolytica exprimiert während seines gesamten Lebenszyklus zwei Cysteinpeptidase-Inhibitoren der Chagasin-Familie (EhICP1 und EhICP2). Beide EhICPs inhibieren die peptidolytische Aktivität von E. histolytica-Zellextrakten und der humanen Peptidasen Cathepsin B und L unterschiedlich effektiv. Innerhalb der Trophozoiten von E. histolytica sind die EhICPs in verschiedenen Kompartimenten lokalisiert, EhICP1 im Cytosol und EhICP2 in Vesikeln, wo es mit verschiedenen lysosomalen Hydrolasen und mit phagocytierten Partikeln kolokalisiert. Im Vergleich zu den amöbialen Peptidasen liegen die EhICPs im molaren Unterschuss innerhalb der Zellen vor. Außerdem ist die Produktion und Lokalisationen der Peptidasen sowie der verschiedenen physiologische Prozesse, an denen die EhCPs beteiligt sind, unabhängig von der Expression der ehicp-Gene. Die erhaltenen Ergebnisse lassen darauf schließen, dass EhICP1 die Zelle vor versehentlich aus undicht gewordenen Lysosomen freigesetzten Peptidasen schützt, während EhICP2 an housekeeping-Prozessen, wie der Kontrolle der Prozessierung von Peptidasen, beteiligt sein könnte. Neben der Charakterisierung der Cysteinpeptidase-Inhibitoren aus E. histolytica bildeten Untersuchungen an einem Annexin-homologen Protein aus Giardia lamblia, einem anderen Intestinalparasiten, einen weiteren Schwerpunkt der hier vorgestellten Arbeit. Das Annexin-homologe alpha-19-Giardin nimmt unter allen Annexinen eine Sonderstellung dahingehend ein, als dass es als bisher einzig bekanntes Annexin ein N-terminales Signal für eine Doppelacylierung besitzt. Mit Hilfe von verschiedenen Expressionssystemen konnte hier experimentell belegt werden, dass alpha-19-Giardin tatsächlich als Substrat für eine Myristoyl- sowie für eine Palmitoyltransferase fungiert, und diese Modifikation die Membranassoziation des Proteins bewirkt.

No description available.

The development of a waterborne pathogen detection method that is rapid, multiplex, sensitive, and specific, would be of great assistance for water treatment facilities and would help protect water consumers from harmful pathogens. Here we have utilized surface enhanced Raman spectroscopy (SERS) in a sensitive multiplex pathogen detection method. Two strategies are proposed herein, one that utilizes SERS antibody labels and one that measures the intrinsic SERS signal of organisms. For the SERS label strategy, gold nanoparticles are conjugated with antibodies specific to Cryptosporidium parvum and Giardia lamblia and with organic dye molecules. The dye molecules, rhodamine B isothiocyanate (RBITC) and malachite green isothiocyanate (MGITC) were surface enhanced by the gold nanoparticles resulting in unique fingerprint SERS spectra. The SERS label method was successful in detecting G. lamblia and C. parvum simultaneously. The method was subsequently coupled with a filtration step to both concentrate and capture cysts on a flat surface for detection. Raman mapping across the filter membrane detected ~95% of the spiked cysts in the optimized system. In the second type of strategy, intrinsic virus SERS signals were detected with silver nanoparticles for enhancement. Principal component analysis performed on the spectra data set resulted in the successful differentiation of MS2 and PhiX174 species and also for the differentiation of viable virus samples and inactivated virus samples. / Ph. D.

Made available in DSpace on 2014-08-20T14:31:31Z (GMT). No. of bitstreams: 1 dissertacao_ana_berne.pdf: 2199093 bytes, checksum: 8e27f9fa1c22e061e919175c81aa8bb3 (MD5) Previous issue date: 2007-03-30 / The enteroparasitosis remains as an important public health problem in children in the Brazil, showing variable prevalence, according to the State and evaluated population. Studies with day-care center children are scarce, however, already knows that the exposure of the children in these places increased the susceptibility to parasitosis. Among the parasitosis the protozoary Giardia lamblia is responsible for severe diarrhea cases in children and the routine diagnosis methods presents many false negative results. The aim of this study was investigate the enteroparasitosis prevalence in children from a day-care public center of Rio Grande county, Rio Grande do Sul State and compare diagnosis techniques in samples of their fecal material to Giardia lamblia , the ELISA immunoassay and the centrifugal-sedimentation methods. 165 fecal samples where evaluated and processed by centrifugal-sedimentation and centrifugal-flotation methods, stained by trichromium and Kinyoun after the concentration by centrifugal-sedimentation. The general prevalence of enteroparasitosis was 64,2% (106/165). The most prevalent nematods species founded was Trichuris trichiura (24,2%) and Ascaris lumbricoides (22,4%) and the the most prevalent protozoary specie was Giardia lamblia (30,3%). The presence of opportunists coccids where also registered Cryptosporidium spp. (2,4%) and Isospora belli (0,6%). Among the positives 56,6% (60 samples) showed simple infection and 43,4% (46 samples) showed associated infection. The presence of non pathogenic protozoary like Entamoeba coli (15,2%), Endolimax nana (3,6%) and Enteromonas hominis (4,8%), indicated environmental fecal source contamination. The higher prevalence of nematods and protozoary in the studied population suggests the necessity of implementation of educational measures to prevent these enteroparasites. In the evaluation of comparative diagnosis of G. lamblia a higher positivity was verified in the ELISA technique 57% (90/158), followed by the centrifugal-sedimentation method 27,8% (44/158). The obtained results in this study suggests that is higher the prevalence of nematods and protozoary in the evaluated children and the ELISA technique to detect antigen in fecal sample showed higher efficiency to giardiasis diagnosis. / As enteroparasitoses ainda constituem um importante problema de saúde pública em crianças no Brasil, com prevalências bastante variáveis, conforme a região e população avaliada. Estudos com crianças que freqüentam creches são escassos, entretanto, sabe-se que nestes ambientes, as crianças estão mais expostas as parasitoses, dentre as quais o protozoário Giardia lamblia que é responsável por quadros graves de diarréia em crianças e os métodos de rotina utilizados no diagnóstico levam a muitos casos de falso-negativos. O objetivo deste trabalho foi investigar a prevalência de enteroparasitos em crianças de uma creche pública do Rio Grande, cidade portuária, localizada na região sul do estado do Rio Grande do Sul e comparar a técnica de ELISA (kit comercial Giardia II) com os métodos de centrífugo-flutuação e centrífugo- sedimentação para o diagnóstico de G. lamblia em fezes de crianças. Primeiramente foram avaliadas 165 amostras de fezes de processadas pelo método de centrífugo-sedimentação e centrífugo-flutuação e pelas colorações de tricrômio e de Kinyoun. A prevalência geral de enteroparasitos foi de 64,2% (106/165). Os nematódeos mais prevalentes foram Trichuris trichiura (24,2%) e Ascaris lumbricoides (22,4%) e o protozoário mais prevalente foi G. lamblia (30,3%). Também foram registradas as presenças dos coccídeos oportunistas Cryptosporidium (2,4%) e Isospora belli (0,6%). Dentre os positivos, 56,6% (60) apresentaram infecção simples e 43,4% (46) associadas. Constatou-se também a presença de protozoários não patogênicos, como Entamoeba coli (15,2%), Endolimax nana (3,6%) e Enteromonas hominis (4,8%), que indicou contaminação de origem fecal do ambiente. A alta prevalência de nematódeos e protozoários na população estudada sugere a necessidade de implementação de medidas educacionais, visando a prevenção destes enteroparasitos. Na avaliação do diagnóstico comparativo de G. lamblia foi verificado maior positividade para a técnica de ELISA, 57% (90/158), seguido do método de centrifugo-flutuação, 30,3% (48/158) e centrífugo-sedimentação, 27,8% (44/158). A partir dos resultados obtidos no presente estudo pode-se concluir que é alta a prevalência de nematódeos e protozoários nas crianças avaliadas e que a técnica de ELISA para detectar antígenos nas fezes é mais eficiente que os métodos de centrífugo-flutuação e centrifugo-sedimentação, podendo, portanto, ser utilizada, tanto no diagnóstico individual como em estudos epidemiológicos da giardíase.

Use of tubular anaerobic digesters to treat livestock waste in developing countries has energy, agricultural, health, social and environmental benefits. However, careful use of digester effluent as a soil amendment is required due to the potential presence of protozoan parasites Cryptosporidium parvum and Giardia lamblia. This research investigated the performance of four tubular digesters in the Monteverde region of Costa Rica. High (>75%) volatile solids and BOD5 removal efficiencies were observed, which was attributed to the formation of a biologically active floccular sludge layer. Computational fluid dynamics (CFD) and bioprocess models were developed to evaluate the transport and transformation mechanisms in the digesters. The CFD model estimated a mean liquid hydraulic residence time (HRT) of 23 days and the bioprocess model estimated an average mean cell residence time (MCRT) of 115 days. Cryptosporidium parvum and Giardia lamblia inactivation studies were performed in the laboratory under conditions similar to the environmental conditions observed in the field tubular digesters. The environmental conditions included: ambient temperatures (21-24°C), neutral pH and total ammonia nitrogen (TAN) concentrations below 250 mg NH4+-N/L. Inactivation rate constants for Cryptosporidium parvum and Giardia lamblia were 0.056 and 0.726 day-1, respectively. An (oo)cysts solid-liquid phase distribution study indicated that 70% of both (oo)cysts adhered to biosolids. A tubular digester model was used to estimate the concentration of viable (oo)cysts in the digester effluents. (Oo)cysts adhesion to solids, total solids concentration in the digester and HRT were the main factors contributing to the modeled effluent concentration of viable (oo)cysts. Since the model predicted presence of viable (oo)cysts in the tubular digester effluent, a quantitative microbial risk assessment (QMRA) model was developed to estimate the risk of infection from exposure to raw livestock waste and tubular digester effluents in two rural communities in Costa Rica. The risk of infection from Cryptosporidium parvum and Giardia lamblia was assessed for occupational and public exposure pathways; fomite and soil contamination and crop contamination from runoff. Results from the QMRA indicated that the concentration of (oo)cysts in the raw livestock waste, inactivation rates at the various exposure pathways and the treatment of livestock waste were the main contributing factors to the risk of infection. This research indicated that treatment of livestock waste in tubular digesters significantly decreased the risk of infection to below WHO’s acceptable individual annual risk of infection (10-4). This is the first study to combine mathematical modeling with field studies to determine the physical and biological processes in tubular digesters. This is also the first study to combine mathematical models with field and laboratory studies to determine the concentration of (oo)cysts in tubular digester effluents and to predict the risk of infection from Cryptosporidium parvum and Giardia lamblia if tubular digester effluent is used as a soil amendment.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-07-17T17:10:52Z No. of bitstreams: 1 Desenvolvimento da PCR em Tempo Real para.pdf: 2139383 bytes, checksum: 1075a6c994ede19cfbf82267ae9a76b9 (MD5) / Made available in DSpace on 2012-07-17T17:10:52Z (GMT). No. of bitstreams: 1 Desenvolvimento da PCR em Tempo Real para.pdf: 2139383 bytes, checksum: 1075a6c994ede19cfbf82267ae9a76b9 (MD5) Previous issue date: 2010 / CNPq e FAPESP / Fundação Oswaldo Cruz. Pavilhão Hélio Peggy e Pereira. Rio de Janeiro, RJ, Brasil / Giardia duodenalis (G. duodenalis) é um protozoário entérico patogênico distribuído mundialmente e apresenta amplo espectro de hospedeiros mamíferos, incluindo humanos. Isolados deste parasito possuem grande diversidade genética, apresentando sete genótipos (A-G) e diversos subtipos. No entanto, apenas os genótipos A e B infectam o homem e no subtipo A1 concentra-se o potencial zoonótico do parasito. Análise das sequências de amplicons de determinados marcadores moleculares demonstrou resultados discordantes na genotipagem de G. duodenalis evidenciando a necessidade de identificar novos marcadores. A proposta desse trabalho foi avaliar a aplicabilidade do locus da enzima málica (ME) comparando os resultados obtidos com o gene da β-giardina (βg) usando a PCR convencional seguida de sequenciamento para detecção e genotipagem de amostras clínicas. Foi desenvolvida uma PCR em Tempo Real - ME para detectar, quantificar e genotipar isolados de G. duodenalis diretamente de amostras de fezes. Foram usadas 100 amostras clínicas (83 humanas e 17 caninas) positivas segundo exame parasitológico e imunológico. A N - PCR convencional - βg amplificou 61% destas (52 amostras humanas e sete caninas) e todas foram caracterizadas como genótipo A, sendo 42 subtipo A1 (38 humanas e quatro caninas) e 19 subtipo A2 (16 humanos e três caninas). A N - PCR convencional - ME detectou apenas nove amostras (9%) positivas e todas pertencentes ao genótipo A, sendo seis humanas (quatro pertencentes ao subtipo A1, uma subtipo A2 e uma com subtipo indeterminado) e três caninas (todas A1). Ao correlacionar a genotipagem por ambos marcadores, observou-se concordância na identificação do genótipo. A comparação do limite de detecção da PCR convencional ME com a PCR em Tempo Real - ME demonstrou que esta última foi aproximadamente 10 4 vezes mais sensível. Análise estatística dos resultados obtidos com as réplicas da curva-padrão indicou reprodutibilidade do método e determinou que amostras negativas são aquelas com valores de Ct > 37. Desta forma, 71 amostras clínicas (71%) foram positivas na PCR em Tempo Real - ME com sonda para subtipo A1, destas 62 (87,3%) eram amostras humanas e nove (13,4%) caninas. A quantificação relativa de DNA de G. duodenalis nas amostras clínicas com a PCR em Tempo Real - ME, demonstrou que a concentração de cistos nas amostras analisadas variou de 4,21 ng a 204 fg (respectivamente, 22.000 e 1,0 cistos). A PCR em Tempo Real - ME apresentou maior sensibilidade em relação à N - PCR convencional - ME, indicando a necessidade de utilização de novos iniciadores, pois os utilizados encontram-se em regiões polimórficas, como demonstrado pela análise das sequências de ME. Apesar da necessidade de mais estudos, os resultados obtidos neste trabalho indicam que a PCR em Tempo Real - ME possui potencial aplicabilidade nos estudos de epidemiologia molecular da giardíase. / Giardia duodenalis (G. duodenalis) is an intestinal protozoan parasite found worldwide in various mammalian hosts, including humans. G. duodenalis isolates display high genetic diversity with seven genotypes (A-G) and subtypes. However, only A and B assemblages have been detected in humans. The subtype A1 parasite presents the strongest zoonotic potential. Discordant genotyping and detection results of G. duodenalis isolates have been previously reported using amplicon sequence analysis from certain molecular markers. Therefore, this leads to the search of novel ones. The purpose of this work was to compare conventional PCR, followed by sequencing, using malic enzyme (ME) and β-giardin gene (βg) as molecular markers for the detection and genotyping of the parasite found in faecal samples. Likewise, an approach involving Real Time PCR - ME for detecting, quantifying and genotyping G. duodenalis isolates was developed. We collected 100 positive faecal samples (83 humans and 17 canines) according to parasitological exam and immunoassays. N - PCR - βg detected 61 positive samples (61%) from which 52 were human and seven were canine. All those samples were characterized as genotype A. Subtype A1 and A2 were determined in 42 (38 humans/4 canines) and 19 (16 humans/3 canines) samples, respectively. However N - PCR - ME analyses revealed that only nine faecal samples (9%) were positive (6 humans/3 canines) for genotype A. Six human samples were subtyped as A1 (4), A2 (1) and one not-determined, and the three canine samples were subtype A2. However, when correlating the sample genotyping using both markers, we have observed an agreement in the identification of the genotypes. The comparison of the detection limit between the N - PCR - ME and the Real Time PCR - ME showed that the latter one was approximately 104-fold more sensitive. The statistical analysis of the data from the standard curve replicates indicated reproducibility of the method. Furthermore, Ct values > 37 were established as an indicative for negative samples. Therefore, Real Time PCR - ME analysis revealed that 71 samples (71%) were positive for subtype A1 from which 62 (87.3%) and nine (13.4%) samples were humans and canines, respectively. The relative quantification of G. duodenalis DNA in the clinical samples using Real Time PCR - ME varied from 4.21 ng to 204.0 fg corresponding to 22.000 and one cyst, respectively. Overall, Real Time PCR – ME analysis showed to be more sensitive than N - PCR - ME. It, thus, suggest the need to design different primers, because the ones used were within a polymorphic region of the ME sequence. Although more investigation is yet to be done, the results achieved in this work indicate that Real Time PCR - ME has a great potential in the applicability for molecular epidemiological studies of giardiasis.

PhD (Microbiology) / Department of Microbiology / Background: Diarrheal diseases have a major effect on human health, Globally; it is second only to pneumonia as a leading cause of death among children under five. They are due to a variety of infectious and non-infectious agents; including Entamoeba spp. Entamoeba histolytica is an invasive enteric protozoan parasite that causes amebiasis. Amebiasis is frequent in communities without clean water and poor sanitation, which include low-income South African populations in Giyani and Pretoria. In these populations, the amount of diarrhea caused by Entamoeba histolytica inclusive of all ages, sexes and HIV status is uncertain. Diagnosis of the parasite is usually by microscopy. However, microscopy lacks sensitivity and specificity, therefore it is not reliable. Fortunately, molecular diagnostic tests have been developed to detect different Entamoeba species in humans. It is known that the parasite E. histolytica causes asymptomatic and symptomatic diseases. However, the transition from colonization to disease is still unclear. While parasite and host factors, as well as environmental conditions influence the infection outcome, there is currently no clear explanation of wide variation in the presentation of the disease. This could suggest that there are other factors affecting the disease outcome. A better understanding of these factors as well as their role in disease remains target objectives of modern scientists and it will definitely help in the fight against the disease. In spite of the emerging evidence that the host microbiome, parasite burden and the inflammatory response contribute to the virulence of E. histolytica, their roles have never been defined in developing regions such as Giyani and Pretoria. In addition, the present study hypothesized that co-infections with E. histolytica and secretion of extracellular vesicles/exosomes have a significant impact on the virulence of E. histolytica. Little has been explored or elucidated about responses triggered by other enteropathogens/ameba interplay that could be important in the induction of tissue invasion and disease and also how E. histolytica/enteropathogens interplay in these infections has not been determined. Therefore, the knowledge of this interplay could help in understanding how this modifies disease manifestations by modulating pathogen virulence and the host response. The use of secretion systems is an essential biological process exploited by pathogenic microorganisms to promote survival and spread of the pathogen, which in turn exacerbate the infection. The study of extracellular vesicles (EVs) released by pathogens is a new and exciting field that may realistically contribute to a better understanding of the pathogenic process of E. histolytica and provide alternate control strategies. Aim and objective of the study: The overall aim of the study was to determine the impact of enteric pathogens and secreted extracellular vesicles on amebic virulence and the outcome of infection. This aim was addressed in through a series of six primary objectives, which were: a. To investigate the distribution and prevalence of protozoan parasites in South Africa. b. To investigate novel species of Entamoeba circulating in the South African population. ix c. To elucidate the impact of gut microbiota and immune response during amebic infection. d. To determine the role of Entamoeba histolytica macrophage inhibitory factor (EhMIF) during amebic infection. e. To investigate the impact of co-infections on the outcome of amebiasis. f. To determine the presence of secreted extracellular vesicles/exosomes in Entamoeba histolytica. Brief methodology and results: A modified and validated Taqman qPCR assay (with taqman probes and genus specific primers) was used for amplification and target detection. This assay was used to investigate the distribution and prevalence of protozoan parasites (Cryptosporidium spp and Giardia lamblia) in South Africa, the assay was considered superior for this project because it is more sensitive than conventional PCR and it can be used to detect multiple infection targets. This assay allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes. A total of 484 stool samples collected from diarrheal and non-diarrheal patients from rural and urban communities of South Africa were studied. The overall prevalence of parasites (Giardia lamblia and Cryptosporidium spp) in rural and urban patients were found to be 49% (112/227) and 21% (54/257) respectively (p= < 0.0001). The distribution of specific pathogens in rural areas was Cryptosporidium spp (20%) and Giardia lamblia (14%). Our findings showed no significant difference in parasitic infections between gender and the age of the participants (Chapter 3). The discovery of novel species is of great importance to human health. We have recently discovered stools positive for Entamoeba organisms by microscopy but PCR negative for known Entamoeba species. This led to the hypothesis that novel species of Entamoeba are present in the South African population. A comprehensive assay was used which included probes to identify Entamoeba bangladeshi from diarrheal and non-diarrheal participants. A sensitive qPCR assays and amplicon sequencing was used to detect Entamoeba spp, Prevotella copri and Enterobacteriaceae. Interestingly, E. bangladeshi was identified in the South African population. Entamoeba was present in 27% (E. histolytica 8.5% (41/484), E. dispar 8% (38/484), and E. bangladeshi 4.75% (23/484) E. moshkovskii was not detected in the present study. We were also able to observe changes in the host microbiome and the parasite burden associated with E. histolytica infections in S. African diarrhea cases versus asymptomatic controls but not with E. bangladeshi or E. dispar. In E. histolytica positive samples the level of both parasite and P. copri were lower in non-diarrheal samples (p=0.0034) (Chapter 4). There is accumulating evidence that the inflammatory response contributes to injury. Little is known about the key parasite mediators of host mucosal immunopathology. This study hypothesized that migration inhibitory factor (MIF) mediates the destructive host inflammatory response seen in amebic colitis. To determine the role of EhMIF during amebic infection, we used a genetic approach to test the effect of EhMIF on mucosal inflammation. We found that EhMIF induces IL-8 secretion from intestinal epithelial cells. Mice treated with antibodies that specifically block EhMIF had reduced chemokine expression and neutrophil infiltration in the mucosa. In addition to antibody-mediated neutralization, mice infected with parasites overexpressing EhMIF had increased chemokine expression, neutrophil influx and mucosal damage. We also found that the concentration of EhMIF correlated with the level of intestinal inflammation in persons with intestinal amebiasis. Together, our results reveal a novel parasite mediator of mucosal inflammation and support MIF homologs as potential immunomodulatory targets (Chapter 5). To investigate the impact of co-infections on the outcome of amebiasis, we analyzed the co-occurence of E. histolytica with other enteropathogens known to cause diarrheal infections, such as Shigella/EIEC (IpaH), Campylobacter (cadf), Enterotoxigenic E. coli (STh), Norovirus GII and Adenovirus (Hexon). The results were compared with those obtained with E. histolytica that were not interacted with enteropathogens and with E. histolytica interacted with enteropathogens. The impact of multiple infections on the outcome of the infection was compared between nondiarrheal and diarrheal stool samples. It was found that co-infections with two pathogens were associated with diarrhea compared to single infections. Moreover, Norovirus GII, Campylobacter (Cadf) and co-infections were associated with diarrhea in the study population. This study did not show any significant impact of pathogens co-infecting with E. histolytica on the outcome of amebic infection (Chapter 6). The presence of secreted extracellular vesicles/Exosomes in Entamoeba histolytica was determined by using the Pathogenic ameba strains (HM-1:IMSS or HM-1:IMSS (Sub-strain-US) from petri’s lab to purify exosomes using the commercially available kit to isolate exosomes (total exosomes isolation kit). Our study for the first time revealed that E. histolytica does secrete Evs. This finding increases the appreciation that all organisms are likely to secrete these EVs (Chapter 7). However, the impact of these EVs on the pathogenesis of E. histolytica needs further investigations. Conclusion: This study has contributed significantly to our knowledge on infectious diarrhea and the diversity of Entamoeba species by providing new data on the rate and prevalence of Entamoeba diarrheal infections and their distribution in the South African population. Our study describes for the first time the presence of E. bangladeshi in the South African population. Furthermore, our results reveal a novel parasite mediator of mucosal inflammation and support MIF homologs as potential immunomodulatory targets. This study also, for the first time revealed that E. histolytica does secrete EVs. The results from this work will undoubtedly open an exciting research to establish a deeper understanding of the function and role of these vesicles in amebic infection. We encourage public health interventions like health education programs and improvement of sanitation and hygiene in these populations. Molecular diagnostics should be used for specific diagnostic in clinical settings. / NRF

Amoebic dysenteri is a problem in developing countries and is caused by Entamoeba histolytica (E. histolytica) with symptoms such as diarrhea, vomiting and in worse case extra intestinal manifestation. Currently there are difficulties to diagnose E. histolytica infections in developing countries because PCR requires advanced and expensive and microscopy cannot distinguish E. histolytica from other harmless species of amoebas. The aim of this study was therefore to develop loop-mediated isothermal amplification (LAMP), which is similar to PCR but is performed at a single temperature and amplifies the target gene in less than an hour. LAMP was also compared to real time PCR. With a commercial kit, DNA were extracted from cultivated trophozoites and for the LAMP reaction, a colorimetric mastermix and six primers were used designed from 18S small subunit ribosomal RNA gene. With phenol red positive LAMP reactions showed a color change from pink to yellow and negative LAMP reactions remained pink. The sensitivity of LAMP for detection of E. histolytica was determined to be 80 pg/µl, which was ten times less sensitive than real time-PCR. The method was also shown to work on trophozoites with no DNA extraction and no non-specific amplifications were seen with DNA from G. lamblia, which showed some specificity. LAMP proved to be sensitive and easy to work with, but requires tightly closed tubes to avoid contamination and false positive results. To develop and evaluate the method LAMP for detection of E. histolytica, more studies are needed, including clinical samples and optimization.