Rozprawy doktorskie na temat „Giardiasis”

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Giardia duodenalis é um protozoário flagelado que coloniza o trato intestinal de hospedeiros vertebrados. A caracterização molecular de G. duodenalis revolucionou a compreensão da taxonomia, diversidade genética e epidemiologia da giardíase em seres humanos e animais. Em nosso estudo, realizamos a caracterização molecular de G. duodenalis em bezerros bubalinos do Estado de São Paulo, Brasil. Assim, foram colhidas 183 amostras fecais de animais da raça Murrah, com até seis meses de idade. Estas amostras foram examinadas por meio da reação em cadeia pela polimerase tipo para amplificação da subunidade menor do gene do RNA ribossômico, todas as amostras positivas por esse gene, foram caracterizadas para amplificação parcial dos genes beta-giardina, glutamato desidrogenase e triosefosfato isomerase. G. duodenalis foi verificada em 6,56% das amostras fecais e por meio da análise das sequências, verificou-se 100% de similaridade genética com “assemblage” E. Esta foi a primeira detecção de G. duodenalis “assemblage” E em bezerros bubalinos no Brasil.
Giardia duodenalis is a flagellated protozoan that colonizes the intestinal tract of vertebrate hosts. A molecular characterization of G. duodenalis revolutionized an understanding of the taxonomy, genetic diversity and epidemiology of giardiasis in humans and animals. In our stud, we performed the molecularly characterization of Giardia duodenalis in buffalo calves from State of São Paulo, Brazil. Then, 183 fecal samples of Murrah buffaloes were collected up to six months of age. These samples were examined by nested polymerase chain reaction for parcial amplification of the small subunit of the ribosomal RNA gene. All G. duodenalis-positive samples were characterized by beta-giardin, glutamate dehydrogenase and triosephosphate isomerase genes. G. duodenalis was detected in 6,56% of the faecal samples, and sequence analysis showed 100% genetic similarity with assemblage E. This was the first detection of G. duodenalis assemblage E in buffalo calves in Brazil.

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O presente trabalho teve como objetivo investigar a ocorrência dos protozoários Cryptosporidium spp. e Giardia spp. em leitões com 45 dias de idade. Amostras fecais de 107 leitões foram colhidas, em três dias alternados, em suinoculturas do Município de Araçatuba, São Paulo, Brasil. A ocorrência de oocistos de Cryptosporidium spp., foi observada em 4,7% (5/107) dos animais usando a técnica de Kinyoun, e a detecção de oocistos de Giardia spp. foi concomitantemente observada em 1,9% (2/107) dos animais pelo método de Faust.. Animais com a presença concomitante de ambos os parasitos apresentaram fezes com aspecto diarréico. A partir dos resultados obtidos é possível inferir que a ocorrência de Cryptosporidium e Giardia foi baixa, o que se atribuiu ao manejo e que a presença destes protozoários não estava associada a todos animais com sintomas diarréicos envolvidos no estudo.
The goal of this research was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in 45-days-old pigs. Fecal samples of 107 pigs were collected at three alternate days in piggeries in Araçatuba, São Paulo, Brazil. Cryptosporidium spp. oocysts were observed in 4.7% (5/107) of animals by means of Kinyoun acid-fast stain and cysts of Giardia spp. were simultaneously observed in 1.9% (2/107) of the animals by the method of Faust. Animals with positivity for the both parasites presented feces with diarrheal aspect. From these results it is possible to infer that the occurrence of Cryptosporidium and of Giardia was low because of the good management practices and both protozoa were not associated the presence of clinical symptoms.

Global Climate Change has empirical evidence to support the idea that CO2 levels may be affecting weather and health, including rates of infectious diseases. The Midwest region of the United States of America has had the highest increase in giardiasis rates in recent years, and Missouri was chosen for this study as a representative state in the Midwest. There is no definitive answer as to why the rates of giardiasis have changed from 2003 - 2013. The Theory of Climate Change was used as the theoretical framework for this study. The purpose of this research was to determine whether temperature, precipitation and CO2 levels are associated with giardiasis. A cross-sectional design was used for this study with a non-probability sample of reported cases of giardiasis for 2003 - 2013, and data were analyzed using a bivariate analysis and multivariate analysis. There was a negative association between precipitation and number of cases of giardiasis in Missouri residents (p < .05), a positive association between temperature and number of cases of giardiasis in Missouri residents (p < .05), and a positive association between CO2 levels and number of cases of giardiasis in Missouri residents (p < .05). Levels of CO2 modified the association between precipitation and number of cases of giardiasis in Missouri residents (p < .05). Levels of CO2 modified the association between temperature and number of cases of giardiasis in Missouri residents (p < .05). These results demonstrate that climatic factors impact public health significantly. The implications for social change are to have the waterways, wells, and public water tested more often, to reinforce the waterway closures with increased measures to prevent morbidity and mortality with giardiasis when possible, and to raise awareness of the climatic impact on health.

The parasites Giardia duodenalis and Cryptosporidium spp., which infect a broad range of vertebrates, are major causes of diarrhoeal disease in humans worldwide. The aim of this thesis was to understand the zoonotic transmission potential of Giardiasis and Cryptosporidiosis in children in Malawi and Cambodia using molecular epidemiological tools. The pattern of faecal Volatile Organic Compounds (VOCs) from children with Cryptosporidiosis in Malawi was also examined. In Chapter 2, faecal samples collected from children under 5 years of age with diarrhoea, living in diverse geographical regions in Malawi, were screened for both parasites microscopically using the Direct Fluorescence Assay (DFA). Nested PCR was used for subtyping Giardia (the bg, tpi and gdh genes) and Cryptosporidium spp. (SSU-rRNA and GP60 genes). The prevalence of G. duodenalis (11.4%,15/132) was significantly lower than the prevalence of Cryptosporidium spp. (23.5%, 31/132) by DFA. In contrast, the prevalence of G. duodenalis (28%,56/200) was significantly higher than the prevalence of Cryptosporidium spp. (11%, 22/200) by nested PCR. Mixed infections with both Giardia assemblage A and B were predominant. The predominance of C. hominis indicated that the anthroponotic route plays a major role of Cryptosporidium transmission in Malawi. In Chapter 3, the prevalence and identity of G. duodenalis assemblages among isolates from children in Cambodia was investigated. Both assemblages A and B were common in Cambodia, however, assemblage B was the most predominant. These data indicate that anthroponotic routes play a major role in Giardia transmission in Cambodia. Nevertheless, Giardia parasites of assemblage B demonstrated higher genetic variation than the other assemblages. Because the major transmission route of Giardia is likely anthroponotic, public health policies should focus on improvements in sanitation and hygiene rather than changes in animal/meat processing. In Chapter 4, I determined genetic variation among Giardia isolates from Malawi and Cambodia. A multiple alignment was performed and phylogenetic trees were generated. The data demonstrated that Giardia assemblage A and B were prevalent in children in both countries, with a predominance of assemblage B. These findings suggest that anthroponotic transmission could be a dominant transmission route for giardiasis in both countries. In Chapter 5, I studied stool samples from patients with confirmed Cryptosporidium infection and negative controls. I tested the hypothesis that the pattern of excreted VOCs could be specific to certain types of diarrhoeal infection. The abundance of several VOCs were significantly different in the two groups; cyclopentane and 3-hydroxy-2-butanone were associated with Cryptosporidium positive samples, and 1-propanol with negative control samples. The presence of cyclopentane and 3-hydroxy-2-butanone and the absence of 1-propanol in Cryptosporidium positive faecal samples could form the basis of a diagnostic test. In Conclusion, my findings suggest that G. duodenalis assemblage B was the predominant assemblage in Malawi and Cambodia. C. hominis was the predominant species in Malawi. Both parasites likely undergo predominantly anthroponotic transmission in these populations. Public health measures targeted at hand washing, improving sanitation, and providing clean drinking water are important strategies for the control of Giardiasis and Cryptosporidiosis infection in these countries.

Resumo: O presente trabalho teve como objetivo investigar a ocorrência dos protozoários Cryptosporidium spp. e Giardia spp. em leitões com 45 dias de idade. Amostras fecais de 107 leitões foram colhidas, em três dias alternados, em suinoculturas do Município de Araçatuba, São Paulo, Brasil. A ocorrência de oocistos de Cryptosporidium spp., foi observada em 4,7% (5/107) dos animais usando a técnica de Kinyoun, e a detecção de oocistos de Giardia spp. foi concomitantemente observada em 1,9% (2/107) dos animais pelo método de Faust.. Animais com a presença concomitante de ambos os parasitos apresentaram fezes com aspecto diarréico. A partir dos resultados obtidos é possível inferir que a ocorrência de Cryptosporidium e Giardia foi baixa, o que se atribuiu ao manejo e que a presença destes protozoários não estava associada a todos animais com sintomas diarréicos envolvidos no estudo.
Abstract: The goal of this research was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in 45-days-old pigs. Fecal samples of 107 pigs were collected at three alternate days in piggeries in Araçatuba, São Paulo, Brazil. Cryptosporidium spp. oocysts were observed in 4.7% (5/107) of animals by means of Kinyoun acid-fast stain and cysts of Giardia spp. were simultaneously observed in 1.9% (2/107) of the animals by the method of Faust. Animals with positivity for the both parasites presented feces with diarrheal aspect. From these results it is possible to infer that the occurrence of Cryptosporidium and of Giardia was low because of the good management practices and both protozoa were not associated the presence of clinical symptoms.
Orientador: Katia Denise Saraiva Bresciani
Coorientador: Marcelo Vasconcelos Meireles
Banca: Fernanda Paiva
Banca: Carlos Noriyuki Kaneto
Mestre

Giardiasis, cause by the parasitic protozoan Giardia duodenalis, is one of the most common infectious gastrointestinal diseases in humans worldwide. However, its true population burden and epidemiology and in particular its zoonotic transmission potential are still poorly understood. Furthermore, G. duodenalis is not a uniform parasite but a complex of seven genetic assemblages or cryptic species (named A to G) that infect humans and a variety of domesticated and wild animals, and that can only be distinguished using molecular genotyping methods. Although there is some evidence that the two Giardia assemblages infecting humans (namely A and B) may differ in their virulence and major transmission routes, data are still scarce. In the UK, several studies suggested that giardiasis is considerably under-diagnosed and a few data are available on the genetic diversity of the parasite causing infection and disease in this country. We investigated the burden, clinical outcomes, risk factors and molecular diversity of giardiasis in North West England using both a descriptive and analytical approach. In Chapter 2, we analysed the self-reported clinical and exposure data collected over four years from clinical cases of giardiasis in Central Lancashire, as part of an enhanced surveillance program on the illness. The resulting average disease rate of 22.5 cases/100,000 population was high when compared to the available national figures. Giardiasis was particularly abundant in adults in their 30s and children under five, and the disease rate in males was significantly higher than in females. Furthermore, the clinical picture of the cases confirmed the high morbidity associated with this infection particularly in terms of the length of illness and severity of symptoms. Only 32% of the cases reported foreign travel during the exposure window. The results suggested the presence of a hidden burden of disease in adults and males, and indicated that local transmission of Giardia can be more common than expected. In Chapter 3, we performed a case-control study to determine the significant risk factors for symptomatic giardiasis in North West England, by recruiting clinical cases of Giardia and age and sex matched controls from Central and East Lancashire and Greater Manchester. The multivariable logistic regression analysis done on 118 cases and 226 controls revealed that overall travelling abroad (particularly to developing countries) was an important risk factor for the illness (OR 9.59). Following the exclusion of participants that reported foreign travel, four risk factors were significant for the acquisition of giardiasis: going to a swimming pool (OR 2.67), changing nappies (OR 3.38), suffering irritable bowel syndrome (OR 3.66) and drinking un-boiled water from the tap (OR 8.17). The results indicated the important role of swimming pools and contact with children in nappies for the transmission of the parasite. In Chapter 4, whole faecal DNA was extracted from the faecal samples of the cases part of the surveillance and case-control studies and the Giardia assemblages and sub-assemblages causing infection were determined using PCR amplification and DNA sequencing of up to four parasite genes (beta-giardin, glutamate dehydrogenase, triose-phosphate isomerase and small-subunit ribosomal RNA). The majority of infections (64%) were caused by assemblage B, followed by assemblage A (33%), whereas mixed-assemblage infections were rare (3%). The majority of the assemblage A isolates belonged to the sub-assemblage AII and showed completed identity with previously described isolates, and six multi-locus genotypes were identified. The level of genetic sub-structuring as revealed by phylogenetic analysis was significantly higher in assemblage B isolates compared with A isolates: a higher proportion of novel assemblage B sequences was detected compared to what was observed in assemblage A isolates. A high number of assemblage B sequences showed heterogeneous nucleotide positions that prevented the unambiguous assignment to a specific sub-assemblage. Up to 17 different assemblage B multi-locus genotypes were found. The molecular genotyping results showed that Giardia assemblage B was responsible for the majority of the clinical infections and confirmed the occurrence of a high diversity of parasite multi-locus genotypes. In Chapter 5, we integrated the epidemiological and the molecular data generated by the enhanced surveillance and case-control studies and we studied the clinico-epidemiological differences between cases infected with Giardia assemblage A or B. Our results showed a difference in the age prevalence between the two assemblages, with assemblage A being more common in older cases. Cases infected with assemblage B reported a series of symptoms more frequently than cases infected with assemblage A, as well as reporting a longer illness. Although the exposure profile of the cases largely overlapped between the two assemblages, two different types of exposures were reported more frequently in the two groups of cases: keeping a dog in assemblage A cases and the presence in the household of children and children at nursery in assemblage B cases. The results suggested that assemblage A could have a major zoonotic reservoir, whereas assemblage B could be transmitted more commonly via the human-to-human route.

In this study the nature and extent of genetic variation in Giardia was investigated to obtain fundamental knowledge on its mode of reproduction, population structure and zoonotic potential, all of which are important in order to fully understand the epidemiology of giardiasis and the taxonomy of the causative agent. Previous studies have contributed little information to these areas due to the comparison of relatively few isolates. Therefore, the major aims of my work were to characterise isolates of Giardia in order to investigate genetic variation and its significance to the epidemiology of giardiasis. Isolates of Giardia. from throughout Australia and overseas, were obtained from humans, cats, cattle, sheep, dogs, goat, beaver and rats and examined using one or more of the following techniques: in vitro cultivation; restriction fragment length polymorphism (RFLP) analysis; enzyme electrophoresis; and amplification of repetitive DNA sequences using the polymerase chain reaction (PCR). In vitro cultivation of Giardia distinguished intraspecific variants based on observed differences in growth between isolates. Human, cat, goat and sheep isolates of Giardia could be established in vitro while isolates of Giardia from dogs and rats failed to establish. Differences in growth behaviour were also observed between isolates of Giardia from the same host species, namely humans and cats. The observed differences in growth during in vitro cultivation in isolates of Giardia from the same and different host species was usually reflected in genetic differences detected by enzyme electrophoresis. Enzyme electrophoresis and RFLP analysis produced very similar groupings of isolates of Giardia with identical banding patterns for all zymodemes and schizodemes, including isolates from restricted geographical areas of Western Australia. The significant correlation between the two techniques suggests that the genetic groups identified by enzyme and DNA analyses consist of natural clones of genetically identical organisms and that Giardia has a clonal population structure. This was supported by the finding of widely distributed common genotypes identified by enzyme electrophoresis throughout Australia and overseas. Electrophoresis banding patterns were of two distinct types. Most isolates produced single-handed enzyme patterns for all the enzymes examined while other isolates produced multiple-banded patterns for a number of enzymes. The observed patterns were interpreted to suggest: (i) that different isolates vary in their ploidy levels with some isolates being functionally haploid and others diploid (or polyploid); and (ii) the possibility of occasional genetic exchange in Giardia. Attempts were made to induce genetic exchange by mixing isolates in vitro and in vivo, but no recombinant genotypes were detected. Enzyme electrophoretic characterisation of isolates of Giardia from different animals and geographical areas, revealed extensive genetic variation with the identification of 47 different zymodemes. The 47 zymodemes could be divided into three main groups: one comprising isolates from humans and a sheep from Western Australia; a second group containing isolates with a worldwide geographical distribution from humans, cats, dogs, sheep, cattle and beaver; and a third group comprising genetically diverse isolates from humans, rats, cat, dog and goat from Australia and overseas. Isolates of Giardia from Aboriginal communities, from within a restricted geographical area, showed a level of genetic diversity similar to that found for isolates characterised from throughout Western Australia. Humans and dogs from these communities were commonly infected with Giardia: PCR analysis of cysts produced similar DNA banding patterns for a number of human and canine faecal samples, which is suggestive of zoonotic transmission. The results of this study show that isolates of Giardia duodenalis are genetically diverse. The source of this diversity is consistent with a predominantly clonal population structure for Giardia with the possibility of occasional bouts of genetic exchange. Further, the identification of genetically similar isolates of Giardia from humans and other animals using molecular techniques, and the extensive genetic diversity detected in isolates from humans, provides evidence for the zoonotic transmission of this parasite.

Метою нашої роботи було вивчення поширеності лямбліозу серед різних вікових груп населення Сумської області. За досліджуваний період з 2004 по 2006 рр., при обстеженні людей різних вікових груп відзначена достовірність до збільшення кількості хворих на лямбліоз у порівнянні з 2001 – 2002 рр. При цитуванні документа, використовуйте посилання http://essuir.sumdu.edu.ua/handle/123456789/5103

Giardia and Cryptospovidium are both recognised as important causes of infectious diarrhoea in children worldwide, and childcare centres have been shown to be a major site of infection. The incidence of infectious diarrhoea in children attending childcare centres has been estimated at between two to five times greater than in children cared for at home. Both Giardia and Cryptospovidium have a faecal-oral route of transmission that facilitates their spread in childcare environments, but can also be interrupted through the use of efficient hygiene protocols such as handwashing. Despite their importance as causes of infectious diarrhoea, there are no data on the prevalence or transmission dynamics of these parasites in Australian childcare centres. The present study was designed to determine the prevalence and incidence of both Giardia and Ciyptosporidium in children attending childcare centres in Perth,Western Australia. Data were collected on asymptomatic infection, seasonal trends, the transmission dynamics of the parasites and risk factors for infection. The second part of the study involved the development, implementation and evaluation of a health intervention package designed to interrupt the transmission of causative organisms of infectious diarrhoea in childcare centres. This intervention was based on appropriate and effective handwashing. Over a period of 23 months, 1 172 faecal samples were collected from non-toilet-trained children (n=306) attending 14 childcare centres in Perth, Western Australia. Where possible, family and contacts of infected children were also sampled to determine the dynamics of infection in the community. Information on syrnptomology of infections and risk factors for infection was obtained by the administration of a questionnaire to parents of all the children in the study. Over all the childcare centres in the study, 7.8% of children were positive for Giardia and 10.8% were positive for Cryptosporidiunz. Of these, 37.5% of the Giardia-positive children returned positive samples on two to four occasions, but not always consecutively, suggesting either continuous or repeated infection with the parasite. Only 12% of children who were Cryptosporidiurn-positive were infected for two consecutive months. The major findings of this study included a significant seasonal peak in the prevalence of both Giardia and Cryptosporidium, with 50% of Giardia and 73% of Cryptosporidiurn infections occurring during the autumn months of March, April and May, and a high proportion of asymptomatic Giardia infections (45%), compared with only 13.5% of asymptomatic Cryptosporidiurn infections. There was evidence for the transmission of both Giardia and Cryptosporidium infections to household contacts of infected children. Of the children who were found to be positive, faecal samples were also collected from 28 family members of those children with Giardia and from 14 family members of those with Cryptosporidiunz. Of these, 17.9% family members of the children with Giardia and 28.6% of family members of the Cryptosporidiuin children returned positive faecal samples. The only significant risk factor for Giardia infection was the number of adults living in the household, with infection more likely to occur in children who had a greater number of adults in the household. Significant risk factors for Clyptosporidium infection included the age of the child: the mean age of the positive children (20.6 months) was higher than in the negative children (16.6 months), and the length of time enrolled at the centre. Children who were positive had attended for a longer time than those who were negative (1 1.2 and 7.8 months respectively). These results have important implications for the control of infection with these organisms, both within and beyond childcare centres. Since these parasites can be readily transmitted by an asymptomatic carrier, the high percentage of asymptomatic cases in this study, particularly of Giardia, strengthens the argument for health interventions which are directed at interrupting the trailsmission of the parasite. A health intervention programme was developed that focused on handwashing procedures and was targeted at the carers, the children and the parents of the children in the centres. It was designed to be a low-cost programme both financially, and in the time and effort required to implement the programme within the childcare centre, to enhance compliance with the intervention. The success of the programme in changing the knowledge, attitudes and practices of carers was evaluated through a pre- and posttest questionnaire. This showed that the programme successfully improved the knowledge of the carers in the test centres in several important areas of infection control. These included knowledge about specific organisms causing infectious diarrhoea in childcare centres, transmission of these by asymptomatic individuals and increased knowledge about effective handwashing technique. Because it has been repeatedly shown that increased knowledge does not always translate into improved practices, and that interventions are not always successful in maintaining an improvement in the desired practices, a subjective evaluation was also performed. This was designed to determine how effective the intervention was perceived to be by the carers themselves, and whether they would continue to use the intervention over time. The results showed that the majority of the carers (>88%) found the intervention appropriate and useful in teaching both the carers and the children within the centres, the importance of handwashing. Twelve months after the intervention had first been implemented, 57% of the centres in the study were still using the intervention at least once per month and a further 2996, while using it less than this, still continued to use it occasionally. This is important information, since an intervention can only be useful if it is actually being used.

Giardia and Cryptospovidium are both recognised as important causes of infectious diarrhoea in children worldwide, and childcare centres have been shown to be a major site of infection. The incidence of infectious diarrhoea in children attending childcare centres has been estimated at between two to five times greater than in children cared for at home. Both Giardia and Cryptospovidium have a faecal-oral route of transmission that facilitates their spread in childcare environments, but can also be interrupted through the use of efficient hygiene protocols such as handwashing. Despite their importance as causes of infectious diarrhoea, there are no data on the prevalence or transmission dynamics of these parasites in Australian childcare centres. The present study was designed to determine the prevalence and incidence of both Giardia and Ciyptosporidium in children attending childcare centres in Perth,Western Australia. Data were collected on asymptomatic infection, seasonal trends, the transmission dynamics of the parasites and risk factors for infection. The second part of the study involved the development, implementation and evaluation of a health intervention package designed to interrupt the transmission of causative organisms of infectious diarrhoea in childcare centres. This intervention was based on appropriate and effective handwashing. Over a period of 23 months, 1 172 faecal samples were collected from non-toilet-trained children (n=306) attending 14 childcare centres in Perth, Western Australia. Where possible, family and contacts of infected children were also sampled to determine the dynamics of infection in the community. Information on syrnptomology of infections and risk factors for infection was obtained by the administration of a questionnaire to parents of all the children in the study. Over all the childcare centres in the study, 7.8% of children were positive for Giardia and 10.8% were positive for Cryptosporidiunz. Of these, 37.5% of the Giardia-positive children returned positive samples on two to four occasions, but not always consecutively, suggesting either continuous or repeated infection with the parasite. Only 12% of children who were Cryptosporidiurn-positive were infected for two consecutive months. The major findings of this study included a significant seasonal peak in the prevalence of both Giardia and Cryptosporidium, with 50% of Giardia and 73% of Cryptosporidiurn infections occurring during the autumn months of March, April and May, and a high proportion of asymptomatic Giardia infections (45%), compared with only 13.5% of asymptomatic Cryptosporidiurn infections. There was evidence for the transmission of both Giardia and Cryptosporidium infections to household contacts of infected children. Of the children who were found to be positive, faecal samples were also collected from 28 family members of those children with Giardia and from 14 family members of those with Cryptosporidiunz. Of these, 17.9% family members of the children with Giardia and 28.6% of family members of the Cryptosporidiuin children returned positive faecal samples. The only significant risk factor for Giardia infection was the number of adults living in the household, with infection more likely to occur in children who had a greater number of adults in the household. Significant risk factors for Clyptosporidium infection included the age of the child: the mean age of the positive children (20.6 months) was higher than in the negative children (16.6 months), and the length of time enrolled at the centre. Children who were positive had attended for a longer time than those who were negative (1 1.2 and 7.8 months respectively). These results have important implications for the control of infection with these organisms, both within and beyond childcare centres. Since these parasites can be readily transmitted by an asymptomatic carrier, the high percentage of asymptomatic cases in this study, particularly of Giardia, strengthens the argument for health interventions which are directed at interrupting the trailsmission of the parasite. A health intervention programme was developed that focused on handwashing procedures and was targeted at the carers, the children and the parents of the children in the centres. It was designed to be a low-cost programme both financially, and in the time and effort required to implement the programme within the childcare centre, to enhance compliance with the intervention. The success of the programme in changing the knowledge, attitudes and practices of carers was evaluated through a pre- and posttest questionnaire. This showed that the programme successfully improved the knowledge of the carers in the test centres in several important areas of infection control. These included knowledge about specific organisms causing infectious diarrhoea in childcare centres, transmission of these by asymptomatic individuals and increased knowledge about effective handwashing technique. Because it has been repeatedly shown that increased knowledge does not always translate into improved practices, and that interventions are not always successful in maintaining an improvement in the desired practices, a subjective evaluation was also performed. This was designed to determine how effective the intervention was perceived to be by the carers themselves, and whether they would continue to use the intervention over time. The results showed that the majority of the carers (>88%) found the intervention appropriate and useful in teaching both the carers and the children within the centres, the importance of handwashing. Twelve months after the intervention had first been implemented, 57% of the centres in the study were still using the intervention at least once per month and a further 2996, while using it less than this, still continued to use it occasionally. This is important information, since an intervention can only be useful if it is actually being used.

El objetivo del estudio fue determinar la prevalencia de Giardia sp. en caninos y niños de comunidades campesinas de los distritos de Ajoyani, Palca y Santa Lucía en Puno. Se recolectaron 130 muestras fecales tanto de niños como de canes. Las muestras fueron conservadas en formol al 10%, siendo luego enviadas al Laboratorio de Parasitología de la FMV-Lima para su procesamiento. Para el diagnóstico de Giardia sp. cada muestra fue analizada mediante las técnicas de Sedimentación Espontánea y Sheather. Considerando como positivo el hallazgo del parásito en una de las dos técnicas usadas, se obtuvo una prevalencia global de 14.6+6.1% y 28.5+7.8% en caninos y niños respectivamente. En caninos se hallaron prevalencias de 31.8%, 18.2% y 9.3% en los distritos de Ajoyani, Palca y Santa Lucía, respectivamente; las prevalencias en machos y hembras fueron de 14.6% y 17.4% respectivamente y según los grupos de edad de 0-6 meses, >6-12 meses, >12-72 meses y >72 meses fueron de 7.7%, 21.7%, 11.4% y 16.0% respectivamente. En niños se obtuvieron prevalencias de 36.4%, 13.6% y 30.2% en Ajoyani, Palca y Santa Lucia, respectivamente; los niños presentaron prevalencias de 14.6% y las niñas 17.4%; según los grupos etarios de 0-3años, >3-7años, >7-12 años fueron de 33.3%, 29.7%, 25.9% respectivamente. Para el análisis estadístico se empleó la prueba de chi cuadrado, con un nivel de significancia de 0,05%. No se encontró asociación estadística significativa (p>0,05) entre la presencia de Giardia sp. y las variables estudiadas. Además la técnica de sedimentación espontánea demostró ser la técnica más eficaz para el diagnóstico del parásito. Las prevalencias halladas en caninos y niños sugieren infecciones independientes. Sin embargo, sólo se esclarecería con futuros estudios moleculares, para descartar posible riesgo zoonótico. Palabras clave: Giardia sp., Puno, Sedimentación espontánea, Sheather, zoonosis
--- The aim of this study was to determine the prevalence of Giardia sp. in dogs and children in rural communities of the districts Ajoyani, Palca and Santa Lucía in Puno. We collected 130 fecal samples from both children and dogs. The samples were preserved in formaldehyde at 10%, then sent to the Laboratory of Parasitology of the FMV-Lima for processing. For the diagnosis of Giardia sp. each sample was analyzed by spontaneous sedimentation techniques and Sheather. Considering as positive the parasite founding in one of two techniques used, we obtained an overall prevalence of 14.6+6.1% and 28.5+7.8% in dogs and children respectively. In dogs, were found prevalences of 31.8%, 18.2% and 9.3% in the districts of Ajoyani, Palca, and Santa Lucía, respectively; the prevalences in males and females were 14.6% and 17.4% respectively and according to age groups from 0-6 months,> 6-12 months, > 12-72 months and > 72 months were 7%, 21.7%, 11.4% and 16.0% respectively. In children, were obtained 36.4%, 13.6% and 30.2% prevalences in Ajoyani, Palca and Santa Lucía, respectively; boys had prevalences of 14.6% and girls had 17.4%, according to the age groups from 0-3years,> 3-7years, > 7-12 years were 33.3%, 29.7%, 25.9%, respectively. For statistical analysis we used the chi square test with a significance level of 0.05%. There was not significant association (p> 0.05) between the presence of Giardia sp. and the variables studied. Furthermore, the spontaneous sedimentation technique proved to be the most effective technique for the diagnosis of the parasite. The prevalence found in dogs and children suggest independent infections. However, only molecular studies would clarify in future to rule out possible zoonotic risk. Keywords: Giardia sp., Puno, spontaneous sedimentation, sheather, zoonosis
Tesis

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Estima la frecuencia de infección con Giardia sp en caninos menores de tres años que concurren a los parques públicos del distrito de Santiago de Surco de Lima Metropolitana, además su asociación con las variables sexo, edad, estado físico de heces, frecuencia de concurrencia a los parques; determina la concordancia entre tres técnicas de diagnóstico. Se recolectan muestras fecales de 140 caninos aparentemente sanos, menores de tres años de edad, los cuales acuden a parques públicos del mencionado distrito y cuyos propietarios acceden participar en el estudio. Para el diagnóstico de las muestras, se utilizan las técnicas de: Inmunoensayo de Cromatografía (IEC), Técnica de Faust (TF) y la Técnica de Sedimentación espontánea (SE), encontrándose frecuencias de 25, 17.9 y 12.1% respectivamente. Al realizar el análisis de asociación entre la frecuencia de infección y las variables predictoras en un modelo de regresión logística no demuestran significancia estadística (p>0.05). Por otro lado, los índices de concordancia de Kappa entre las pruebas diagnósticas son 0.78 (IEC versus TF), 0.58 (IEC versus TSE) y 0.77 (TF verss TSE), demostrando todas significancia estadística (p<0.05). Estos resultados indican un moderado nivel de infección con Giardia sp. en caninos que concurren a parques públicos en el distrito de Santiago de Surco-Lima, evidenciando un potencial riesgo zoonótico para la población humana. Adicionalmente, tanto la IEC como TF demostraron un nivel de concordancia substancial.
Tesis

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Brinda información actualizada sobre la epidemiología, diagnóstico, tratamiento, prevención y control de la giardiasis en caninos y felinos, que pueda ser aplicada por el Médico Veterinario en nuestro medio. En la actualidad se reconocen 6 especies de Giardia, con distinta especificidad de hospederos. Además, se conoce que Giardia duodenalis posee una gran variabilidad genética, presentando un conjunto de genotipos morfológicamente indistinguibles que pueden afectar a diversos mamíferos e incluso al hombre. Los diagnósticos utilizados con mayor frecuencia son los métodos microscópicos tradicionales, existiendo otras técnicas de valor epidemiológico como ELISA y otra de mayor sensibilidad y especificidad como la técnica de inmunofluorescencia directa; el uso de las técnicas moleculares como PCR, han permitido identificar los genotipos existentes. El tratamiento de la giardiasis debe ser efectivo e integral y requiere la aplicación de medicamentos seguros, los cuales deben pertenecer a los grupos químicos de los benzimidazólicos, nitroimidazoles, nitrofurano y la quinacrina; los cuales requieren de una aplicación prolongada, que varían, entre 2 a 10 días. Sin embargo, la eficacia de alguno de estos medicamentos hasta la fecha no ha alcanzado el 100%; debido a que el período prepatente de la Giardia sp., puede ser extremadamente corto; pudiendo ser posible que el animal se reinfecte e inicie una nueva eliminación de quistes luego de cinco días contados a partir del último tratamiento. Por lo que es muy difícil prevenir la reinfección de mascotas caseras o de animales que se conservan en ambientes no controlados. Además, se requiere de la aplicación de medidas de vigilancia tendientes a la disminución de la carga infectiva (quistes) presentes en los lugares de recreo y en los pelos de los caninos y felinos, realizando una descontaminación del ambiente en base a amonio cuaternario y baño de la mascota; evitándose de esta forma la continuación del ciclo biológico.
Trabajo de suficiencia profesional

In this study, we determined the immunogenicity of CWP2 and its potential as a vaccine candidate against giardiasis. CWP2 was expressed as a recombinant protein with an hexa-histidine affinity tag and was isolated from inclusion bodies. When BALB/c mice were immunized with CWP2, a specific IgA was detected in the feces. When mice were immunized with CWP2 + cholera toxin, as an adjuvant, IgA in the feces, and IgA, IgG1, and IgG2a in the serum, all specific to CWP2, were detected. Also, CD-1 mice were infected with G. muris and presence of specific IgA antibodies to CWP2 were detected in the feces. This result indicated that CWP2 was recognized by the immune system in a natural infection. IL-4 and IL-5 were released from Peyer's patches (PP) and mesenteric lymph nodes (MLN) cells when stimulated with concanavalin A. In spleen cells, IFN-gamma, IL-4, and IL-5 were released when stimulated with concanavalin A. However, in PP, MLN and spleen cells, the levels of cytokines were barely detectable when stimulated with CWP2. The presence of IgG2a (Th1), IgA and IgG1 (Th2) as the production of IFN-gamma (Th1), IL-4 and IL-5 (Th2) confirmed that CWP2, when presented orally to mice, stimulates both a Th1 and Th2 type immune response, locally and systemically.

Background: Giardiasis is one of the most common causes of diarrheal disease worldwide and 5-nitroimidazoles (5-NI) are the most commonly prescribed drugs for the treatment of giardiasis. We evaluated the efficacy of 5-nitroimidazoles (5-NI) in the treatment of giardiasis in a systematic review of randomized controlled trials (RCTs). Methodology/Principal Findings: We conducted a comprehensive literature search in PubMed-Medline, Scopus, Web of Science and Cochrane Library for RCTs evaluating the efficacy of 5-NI vs. control (placebo or active treatment) on parasitological cure in patients with parasitologically-demonstrated giardiasis. The search was performed in May 2013 with no language restriction by two authors independently. The efficacy outcome was parasitological cure, and harmful outcomes were abdominal pain, bitter or metallic taste, and headache. We included 30 RCTs (n = 3,930). There was a significant and slightly higher response rate with 5-NI in giardiasis treatment (RR 1.06, 95%CI 1.02–1.11, p = 0.005). There was high heterogeneity among studies (I2= 72%). The response rates for metronidazole, tinidazole and secnidazole were similar (RR 1.05, 95%CI 1.01–1.09, p = 0.01; RR 1.32 95%CI 1.10–1.59, p = 0.003; and RR 1.18 95%CI 0.93–1.449, p = 0.18, respectively). On subgroup analyses, the response rates did not vary substantially and high heterogeneity persisted (I2= 57%–80%). Harmful outcomes were uncommon, and 5-NIs were associated with lower risk of abdominal pain, and higher risk of both bitter or metallic taste and headache. Conclusions: Studies investigating the efficacy of 5-NI in giardiasis treatment are highly heterogeneous. 5-NIs have a slightly better efficacy and worse profile for mild harmful outcomes in the treatment of giardiasis in comparison to controls. Larger high quality RCTs are needed to further assess efficacy and safety profiles of 5-NI.

The straw-necked ibis (Threskiornis spinicollis) is a large wading bird of the family Plataleidae, subfamily Threskiomithidae, endemic to Australia and Papua New Guinea. It breeds in large, noisy colonies, often numbering in thousands to tens-of-thousands of breeding pairs. This thesis was undertaken to investigate sickness and mortality occurring in ibis nestlings at two colonies in Western Australia (Capel, approximately 300 km south of Perth, and Cataby, 160 km north of Perth). A multi-disciplinary approach was taken. A detailed post-mortem and clinical investigation was conducted (gross pathology, bacteriology, toxicology for selected pesticides and heavy metals, parasitology, haematology, biochemistry, and limited virology using scanning electron microscopy). As a result of these investigations, Giardia, a flagellate protozoan known to cause disease in many vertebrate species, was the only consistent pathogen recognised. Toxicological studies were negative, and haematological and biochemical results inconclusive. As such, cohorts of nestlings were monitored over time for prevalence of Giardia infection, weight gain, and survival. Histological studies and statistical analyses were used to determine whether or not Giardia was responsible for sickness and death in the ibis nestlings. The progress of infected birds removed from the wild and held in captivity was also monitored. As a first step in determining if infection might be a threat to other avian species, Giardia prevalence in other wild bird species was investigated. In an effort to better understand the nature of isolates obtained, they were characterised using scanning electron microscopy and multi-locus enzyme electrophoresis, then compared with avian isolates described by other researchers. A detailed clinical and post-mortem investigation was conducted during the 1993 breeding season. Giardia trophozoites were detected histologically in the duodenums of seven of 28 birds examined, but there was no histological evidence of pathogenicity. Twenty-six of the 28 birds had trophozoites in scrapings of duodenal contents. Blood smears from nine of 25 birds examined exhibited numerous immature heterophils, a condition that may have been indicative of serious disease; however, no cause could be found and the condition was not associated with Giardia infection or ill health. Further, Giardia infection was not associated with weight loss or death. During the 1993 breeding season, the mortality rate of birds wing-tagged at approximately two to three weeks of age and monitored to fledging (seven to nine weeks of age) was found to be 48% at Capel and 83% at Cataby. During three breeding seasons (1990 to 1993), the overall prevalence of Giardia infection of young ibis at both sites was 74%. Prevalence was significantly higher at Cataby than at Capel (χ21= 5.76, P< 0.05), and was significantly higher at Capel in 1992 than in 1991 (χ21= 6.67, P < 0.05), although these results may have been influenced by the different ages at which birds were sampled. The proportion of infected birds at Capel increased from 1991 to 1993 (χ21= 4.72, P < 0.05). This work indicated that Giardia infection is probably endemic in these populations and that prevalence varies between sites and seasons. Sick birds removed from the wild thrived in captivity and were successfully released. Treatment with dimetradazole appeared to clear the birds of giardial infection, with no cysts being evident in faeces later than one week after treatment. The survey conducted for the presence of Giardia included species of birds nesting with infected ibis, and other species collected near Perth. One-hundred and forty-eight individual birds, representing 39 species, were examined. Nine birds. representing two orders and four species, were positive for Giardia'. one rufous night heron (Nycticorax calendonicus) (n = 1); four great egrets (Egretta alba) (n = 9); three sacred ibis (Threskiornis aethiopica) (n = 7); and one Pacific black duck (Anas superciliosa) (n = 12). All positive samples came from living birds at the Capel site. with the exception of one from a sacred ibis collected in suburban Perth. These results suggest that Giardia infection is not widespread among bird taxa in Western Australia; but that ciconiiforms are principal carriers of Giardia in wild bird populations, as appears to be the case in North American situations. The survey results also suggest that infection with Giardia among wild birds in south-western Australia may be confined to particular wetlands, and that ardeids such as the great egret (Egretta alba) nesting near infected straw-necked ibis may also have high infection rates. Attempts were made to adapt a commercially-available enzyme-linked immunosorbent assay (ELISA), used for the detection of Giardia antigen in human faeces, for antigen detection in ibis faeces. The ELISA achieved a 100% specificity. but sensitivity was poor and was thought most likely to be a reflection of a low concentration of antigen in the ibis faecal samples. Scanning electron microscopy (SEM) and multi-locus enzyme electrophoresis (MLEE) performed on the ibis isolates showed them to have identical enzyme profiles and morphology, suggesting that one strain was infecting birds at both colonies. Comparison with G. ardeae, isolated in the U.S.A. from the great blue heron, Ardea herodias, (Erlandsen et al., 1990), revealed distinct enzyme profiles and morphology, although the ibis strain shared some bands and most morphological characteristics with G. ardeae. Comparison with isolates of Giardia obtained from humans (G. duodenalis) revealed a very different enzyme profile and morphology. This suggested that the ibis-derived isolates may have no zoonotic potential; although humans can be infected with genetically different strains, isolates similar to G. ardeae have not been recovered from humans. It was concluded that one strain of Giardia was infecting birds at both sites. that it was probably closely related to G. ardeae, and was not likely to possess zoonotic potential. Giardia was highly prevalent in ibis nestlings, but, as a sole factor, did not appear to be causing the deaths observed. However, because it was the only potential pathogen consistently identified, the role of Giardia infection in conjunction with some other factor or factors should be further investigated.

Biological and immunological aspects of the host-parasite relationship were examined in mice which are susceptible (A/J) and resistant (B10.A) to Giardia muris. B10.A exhibited a shorter latent period, lower cyst output during the acute phase of the infection and shorter period of cyst release compared to A/J. Characteristics of the infection transmitted from mouse-to-mouse and those induced by oral inoculation with cysts or trophozoites were similar. The infection was longer in male A/J and B10.A mice compared to females. Susceptibility and resistance during both the acute and elimination phases of the infection were under non-H-2-linked multigenic control. A/J and B10.A differed in non-specific serum IgG and IgA, but not in the specific IgG and IgA to G. muris. Specific antibodies participated in complement-mediated killing of trophozoites. Spleen, mesenteric lymph node and peritoneal cells from A/J and B10.A mice had a similar ability to kill trophozoites. The capacity of B10.A to mount inflammatory responses was greater than that of A/J. A/J were more immunosuppressed than B10.A during the infection, particularly at mucosal sites. Macrophage-like suppressor cells were shown to be the mediators of this suppression.

Gerbils, experimentally infected with Giardia lamblia trophozoites, had trophozoites and encysting trophozoites in all 3 equal sections of the small intestine and in the colon at necropsy. Cysts were found in the second and third sections of the small intestine and in the colon. WB strain derived trophozoites (WB, D1, WB-C6, and V1) differed in levels of encystation in vitro but not in gerbils. Passage in gerbils increased the in vitro encystation levels of WB and D1 but decreased that of WB-C6 and V1. No differences were found in the total protein profiles or isoenzyme patterns of these G. lamblia populations. Immunization of mice with in vitro cysts produced monoclonal antibodies (mAbs) recognizing cyst protein antigens. In Immunofluorescence (IFA), mAb 5A4.G6 recognized cyst walls. This mAb reacted with a 38 kD band on Western blots. IFA results showed that mAb 8C5.C11 reacts with vesicles in encysting trophozoites and with cyst walls. It recognized 26, 28, 42 and 46 kD bands in Western blots. When mAb 8C5.C11 and Guinea pig complement were added to 0-9 hour encysting cultures, the numbers of cysts produced were significantly reduced compared to control. MAb 5A4.G6 did not affect in vitro encystation.

Orientador: Anete Pereira de Souza.
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Giardia duodenalis é um protozoário flagelado que parasita o homem e diversos animais domésticos e selvagens. Este parasito causa a doença giardiose que é uma das mais prevalentes doenças parasitárias de veiculação hídrica do mundo, responsável por aproximadamente 280 milhões de casos anualmente. Existe uma considerável variabilidade genética em G. duodenalis, de modo que seus isolados foram divididos em oito grupos genéticos (A-H), dois dos quais (A e B) são encontrados tanto em humanos quanto em animais. Os demais grupos (C-H) parasitam outros animais e apresentam maior especificidade a determinados hospedeiros não humanos. A contaminação ambiental por Giardia tem sido amplamente descrita embora esses estudos, em sua maioria, são realizados no nível de identificação de espécie. Há falta de estudos que correlacionam a contaminação ambiental e infecções clínicas na mesma região. O presente trabalho teve como objetivo principal contribuir para o conhecimento da diversidade genética da espécie Giardia duodenalis. Primeiramente, foi realizada a genotipagem multilocos dos principais grupos genéticos de G. duodenalis na região metropolitana de Campinas. Foram encontrados grupos genéticos associados principalmente a infecções humanas bem como isolados com potencial zoonótico em amostras ambientais e obtidas de outros animais. Foi encontrado um alto percentual (25%) de amostras com grupos genéticos mistos e um elevado número de haplótipos distintos, indicando grande diversidade genética do parasito nessa região. Na segunda parte deste trabalho, foi realizado um estudo populacional com amostras clínicas de Giardia provenientes de hospital, creche e centro de controle de zoonoses e amostras ambientais de esgoto hospitalar, efluente de estação de tratamento de esgoto e amostras hídricas de importantes rios e córregos urbanos. As análises populacionais, com exceção das amostras caninas, evidenciaram grande similaridade genética entre essas populações de Giardia. Na terceira parte do presente trabalho, foi realizada uma busca por repetições microssatélites (SSRs) nos genomas publicados de Giardia para desenvolvimento, caracterização e avaliação de polimorfismo de novos marcadores microssatélites. Foram encontrados 506, 438, 402 e 507 microssatélites correspondentes aos genomas AI, AII, B e E, respectivamente. Foram selecionados 80 SSRs específicos aos grupos genéticos A, B e E (40, 20 e 20, respectivamente), além de 36 SSRs compartilhados entre os três genomas. A análise de amplificação confirmou a existência de marcadores específicos aos grupos genéticos A, B e E, além de marcadores compartilhados entre os grupos. A caracterização dos SSRs permitiu a detecção de 12 locos SSRs polimórficos do grupo genético A e sete locos SSRs polimórficos do grupo genético B. Dentre os marcadores compartilhados, o loco GduABE01 apresentou polimorfismo. Os locos polimórficos podem servir para futuros estudos populacionais e os marcadores desenvolvidos podem ser utilizados para identificação dos principais grupos genéticos de G. duodenalis em amostras clínicas e ambientais. Os resultados apresentados contribuem para um melhor entendimento sobre a diversidade genética do parasito bem como sobre a presença de grupos com potencial zoonóticos inter-relacionados em diferentes regiões. Os novos marcadores moleculares disponibilizados podem contribuir para novos estudos populacionais, promovendo melhor discriminação entre os genótipos e possibilitando assim identificar a contaminação e promover o rastreamento da doença
Abstract: Giardia duodenalis is a flagellate protozoan that that parasites humans and several domestic and wild animals. This parasite causes giardiasis, one of the most common waterborne diseases in the world responsible for, approximately 280 million cases per year. There is a great genetic diversity in this species and its isolates have been grouped into eight distinct genetic assemblages (A-H). While groups A and B parasitize different hosts and have zoonotic potential, groups C, D, E, F, G and H usually found in animals and show greater specificity to the parasitized host. Environmental contamination for Giardia has been widely reported however, most of these studies have been performed only at species level. The present study aimed to contribute to the knowledge of the genetic diversity of the species Giardia duodenalis. In the first chapter of this document, multilocus sequence-based genotyping using three gene loci assigned most of the samples as belonging to human genotypes although isolates with zoonotic potential have also been identified in environmental and non-human clinical samples. A high percentage (25%) of mixed assemblages and a high number of different haplotypes were detected, which indicates high genetic diversity of this parasite in this region. In the second chapter, a population genetics study was performed with clinical samples from hospital, day-car center and a center for zoonosis control of the city and environmental samples from hospital sewage, effluent of a wastewater treatment plant and important water samples from rivers and urban streams. With the exception of the canine population, population genetic analysis showed consistent similarity between clinical and environmental populations. In the last chapter, we performed a search for microsatellites (SSRs) in the published genomes of Giardia to develop and characterize the polymorphism of new microsatellite markers. Our group identified 506, 438, 402 and 507 microsatellites of the genomes AI, AII, B and E, respectively. We have selected 80 markers specific to the genetic assemblages A, B and E (40, 20 and 20, respectively) and 36 shared SSRs between the three genomes. Analysis of amplification reactions confirmed the existence of specific loci of each genetic assemblage as well as shared loci among assemblages. Characterization of all loci allowed the detection of 12 polymorphic loci for group A and seven polymorphic loci for group B. Among the shared markers, GduABE01 presented polymorphism. The polymorphic markers can be used in future population genetic studies and the developed markers can contribute to the identification of the main genetic assemblages of G. duodenalis in clinical and environmental samples. The results presented here contribute to a better understanding of the genetic diversity of the parasite as well as the presence of zoonotic potential genotypes, related in different regions. The new molecular markers provided can contribute with population genetic studies in a high level of discrimination that allows identifying the source of contamination and molecular tracking of the disease
Doutorado
Genetica de Microorganismos
Doutor em Genetica e Biologia Molecular

In mice infected with 10$ sp4$ Giardia muris cysts, a peak lymphocyte proliferation in the spleen and Peyer's patches in response to Giardia extract occurred during the elimination and latent phases, respectively. This shows that the Peyer's patch cells are more responsive than the spleen to Giardia infection. Th2-type cytokines produced by Peyer's patch cells may play a protective role during the latent and acute phases. Th1-type cytokines may contribute to this production during the elimination phase. Cytokine production in response to Giardia extract in vitro was observed in mice immunized with this extract, but not in control mice. Therefore, Giardia antigen can induce cytokine production in vitro in a specific manner.

Giardia is the most common human parasite infection in the United States causing a lengthy diarrhea. Transmission of Giardia is by the fecal-oral route and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia in drinking water through the "Surface Water Treatment Rule." Current methods for detection of Giardia in water rely primarily on microscopic observation of water concentrates by immunofluorescent techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 base pairs long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of M13 vector with insert was isolated from lysed host E. coli XL1- Blue and used for production of the cDNA probe by nick translation with ³²P-labeled nucleotides. Seven different protocols were tested for extracting nucleic acids from the cysts. Using the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates by dot-blot hybridization assays. Environmental concentrates from secondary and tertiary treated sewage or surface waters were screened for Giardia cysts by immunofluorescent and the genespecific probe. Positive signals were observed in sewage and surface water samples without floatation at ten fold greater dilutions than after floatation. It appeared that gene probe detection was slightly more sensitive than microscopic detection of Giardia cysts for wastewater samples. In six surface water samples and two sewage sample no positive results were found either by the cDNA probe or immunofluorescent. Usually, DNA probes are radiolabeled and the most commonly used is ³²P. ³²P is expensive, hazardous and has an extremely short half-life of 14.3 days, necessitating frequent preparation of the nucleic acid probes. Three non-radioactive labeling methods, chemiluminescence, enzyme-linked immunoassay and enhanced chemiluminescence were evaluated. The cDNA probe was labeled by nick translation for chemiluminescence method. Biotinylated deoxyuridine triphosphate was used in place of deoxythymidine triphosphate to produce biotinylated DNA strands. The result of hybridization was visualized by chemiluminescenct detection of DNA. The sensitivity of the chemiluminescent method and the 32P labeled probe was 0.1 pg of DNA in a slot-blot hybridization assay.

Studies using Mongolian gerbils found that during a primary infection with Giardia lamblia trophozoites, disaccharidase activities were decreased from day 10 post-infection (p.i.) until well past elimination of the parasite. However, during a challenge infection, enzyme deficiencies were short-lived. A challenge with a soluble extract of G. lamblia trophozoites also resulted in reductions in disaccharidase activity. The degree of these reductions in enzyme activity was dependent on the extract dose. Gel filtration of the trophozoite crude extract resulted in fractions F1, F2, and F3. However, only a challenge with F1 led to disaccharidase deficiencies. Further separation of F1 resulted in fractions F1a and F1b. Impairments of enzyme activity were obtained only in gerbils challenged with F1b. Protein analysis of F1b revealed several high and low molecular weight bands. When gerbils previously exposed to G. lamblia were challenged with an extract of Entamoeba histolytica trophozoites, disaccharidase activities remained comparable to controls. Moreover, enzyme levels in gerbils challenged with excretory/secretory G. lumblia products were affected in a manner which was inconsistent with the live parasitic challenge. Results suggest that the disaccharidase deficiencies in giardiasis are parasite-specific and are induced by a heat-stable constituent(s) of fraction F1b, possibly through an immune response to an antigenic component of this parasite fraction.

Evalúa los parámetros de tiempo, temperatura y concentración de antimicrobianos para el crecimiento de Giardia lamblia en medio de cultivo TYI-S-33 comercial y artesanal. Se llevó a cabo en el Laboratorio de Enteroparásitos de la DEET, del Centro Nacional de Salud Pública del Instituto Nacional de Salud. Se trabajó con la cepa de Giardia intestinalis ATCC 30957 criopreservada a -70°C procedente de un aspirado duodenal de un paciente varón y se reactivó en medio TYI-S-33 comercial, las resiembras se realizaron en medio TYI-S-33 comercial y artesanal, que contenían los antibióticos; penicilina (394 U/ml), estreptomicina (394 μg/ml), gentamicina (50 μg/ml) y amikacina (156 μg/ml), para comparar la carga parasitaria en cada uno de ellos y determinar el mejor medio de cultivo para su desarrollo. Así mismo, se determinó la curva de crecimiento durante 216 horas a tres temperaturas distintas (36°C, 36.5°C y 37°C) determinándose que Giardia lamblia tiene un mejor crecimiento en medio de cultivo TYI-S-33 artesanal a 37°C y una fase log entre las 72 a 120 horas.
Tesis

Лямбліоз – антропонозна протозойна хвороба з переважним функціональним порушенням дванадцятипалої кишки та інших органів травлення, яке у більшості людей перебігає безсимптомно. У розвинутих країнах інвазування лямбліями серед дорослого населення складає близько 5 %, а в розвиваючих понад 15 %. В Україні інвазованість лямбліями серед дорослого населення з патологією щлунково-кишкового тракту складає 2 %. Актуальність полягає в тому, що лямблії сприяють токсико-алергічну дію на весь організм людини, призводять до змін у роботі нервової системи (нейроциркуляторна дистонія), серцево-судинної системи (функціональна кардіоміопатія) та найбільший вплив Lamblia intestinalis має на шлунково-кишковий тракт. Рух лямблій порушує цілісність слизової оболонки тонкої кишки, що в поєднанні з токсико-алергічним ефектом призводить до ентериту.

Giardia intestinalis is the most common parasite found in dogs and cats. It is traditionally diagnosed using a microscope. These methods include direct immunofluorescence, DIF, and zinc sulphate centrifugal flotation, ZnSO4 C-flotation. However, there are commercially available SNAP tests such as the FASTest® GIARDIA strip that is often used by dogs and cats owner to detect Giardia. The aim of this study was to compare the sensitivity, cost and labor intensity of these three methods for detection of Giardia. To investigate this, 150 samples from dogs and cats were examined at the National Veterinary Institute in Sweden. The samples were a mixture of diarrheic and non-diarrheic stool. Of the 150 stool samples 100 samples were examined with FASTest® GIARDIA strip while 150 samples were examined with DIF and ZnSO4 C-flotation. The results indicated that FASTest® GIARDIA strip had a sensitivity of 66.18 %, a cost of 100 Swedish crowns (SEK) per sample and was the easiest test to use. ZnSO4 C-flotation had a sensitivity of 89.90 %, cost 418.75 SEK and took about 15 minutes to perform. DIF had 100 % sensitivity and specificity and due to that it was used as a standard reference method. The cost for DIF was 300 SEK and took more than an hour to perform per sample. The conclusion from this study is that, FASTest® GIARDIA strip is not a recommended test for detection of Giardia despite their low cost and easiness to use. DIF and ZnSO4 C-flotation remain a better diagnostic option for detection of Giardia.

Publicación a texto completo no autorizada por el autor
Determina la prevalencia de giardia spp. y Cryptosporidium spp. en caninos criados en los distritos del Cono Oeste de Lima Metropolitana. Además, evaluar su asociación con las variables: sexo, edad, estado físico de las heces, tipo de alimentación y permanencia en el hogar. Se recolectaron 300 muestras fecales de perros aparentemente sanos, de ambos sexos, diferentes razas y edades comprendidas entre 1 mes a 12 años. Se utilizó las técnicas de sedimentación espontánea, para el diagnóstico de Giardia spp. y la tinción de Ziehl-Neelsen modificado, para Cryptosporidium spp.; encontrándose prevalencias de 16.7±4% y 29.7±5%, respectivamente. Mayor porcentaje de Giardia spp. presentaron los animales menores de 6 meses que los mayores de 6 años; mientras que animales menores de uno y mayores de 6 años, mostraron frecuencias altas en Cryptosporidium spp. Encontrándose asociación significativa entre la edad y la presencia de estos parásitos. Así mismo, formas parasitarias de ambos protozoos fueron detectados con mayor frecuencia en heces sueltas que en las normales, existiendo diferencias significativas en Giardia spp. no así en Cryptosporidium spp. No se hallaron diferencia significativa entre la presencia de Giardia spp. y/o Cryptosporidium spp. con el sexo, tipo de alimentación y permanencia en el hogar de los canes. Estos resultados, demostrarían la existencia de una prevalencia moderada de Giardia spp. y Cryptosporidium spp. en la población canina de una importante zona urbana de Lima y su presencia en caninos podría constituir un serio problema para la Salud Pública, en especial a niños y personas inmunosuprimidas.

Giardia are a genus of enteric pathogens consisting of at least six species (Monis et al. 2009), of which one, Giardia duodenalis, infects humans (Heyworth 2016). In addition to humans, G. duodenalis parasites infect other mammals, which may act as reservoirs for human infection (Traub et al. 2004; Yaoyu and Xiao 2011; Abeywardena et al. 2015; Sroka et al. 2015; Štrkolcová et al. 2015; Heyworth 2016). In humans Giardia infection can be asymptomatic, however all infected hosts shed cysts and can transmit parasites (Oliveira-Arbex et al. 2016; Figgatt et al. 2017). Giardia infection can cause giardiasis, a diarrhoeal disease with a variety of clinical manifestations (Wolfe 1992; Homan and Mank 2001; Sahagún et al. 2008; Nielsen et al. 2014). An estimated 180 million symptomatic human Giardia infections occur every year (Kirk et al. 2015), and treatment is reliant on a small number of chemotherapeutic classes, all of which are associated with liabilities. Liabilities include but are not limited to; poor treatment efficacies, long treatment courses and side-effects which impact compliance (Cina et al. 1996; Wright et al. 2003; Escobedo and Cimerman 2007; Nabarro et al. 2015). Growing parasite resistance to the first-line treatment drugs, the 5-nitroimidazoles, is also a concern (Nabarro et al. 2015). The aim of the current study was to pave the way towards improved treatment options for giardiasis by identifying new lead compounds for drug development, and to further examine the activity of these compounds. To achieve this, a new anti-Giardia activity assay to assess parasite growth in micro-titre plates under microaerobic (3 % O2) conditions was developed. This image-based assay uses bright-field microscopy paired with digital phase-contrast microscopy and supervised machine learning software, PhenoLOGIC and Harmony® (Perkin-Elmer, USA) to differentiate and enumerate parasites. Growth assessment does not require cell-staining or a genetically modified parasite line, thus it can assess the growth of any established Giardia line, at multiple time-points which are distinct advantages over other assays currently used in the Giardia field. Importantly, this assay gives 50 % inhibitory (IC50) values for control compounds metronidazole (IC50; 2.7 ± 0.7 µM), albendazole (54 ± 5 nM) and furazolidone (200 ± 90 nM) , against BRIS/91/HEPU/1279 at 48 h consistent with those previously reported by others (Edlind et al. 1990; Cedillo-rivera et al. 2002; Hounkong et al. 2011; Tejman-Yarden et al. 2011). The validated image-based assay was used to screen a sub-set of Compound Australia’s Open-access Scaffold Library for anti-Giardia activity. A total of 2451 compounds (two per scaffold) were screened at 10 µM. Forty-one compounds (1.7 % hit rate) were validated as having anti-Giardia activity (>50 % inhibition at 48 h) in these assays. Secondary testing of hit compounds to determine IC50 values against Giardia and neonatal foreskin fibroblasts (NFF) identified five compounds with IC50 values <1 µM and >10 fold selectivity for parasites over mammalian cells. Rational selection based on selective activity, chemical novelty and chemical liabilities identified seven hit series for further investigation. Compounds within these series (196 total; ~28/series) were then assessed to examine structure activity relationships (SARs) and prioritize hit series for development. Analogues of particularly potent and selective series were also synthesized by collaborators and assessed for anti-parasitic activity and selectivity. The most promising hit was three orders of magnitude more potent than the current first-line anti-Giardia treatment drug, metronidazole (SN00798527; series CL9569; 48 h IC50 9 nM vs. metronidazole 48 h IC50 3 µM), with a selectivity index (SI) of >11,000. Importantly, this activity was maintained against multiple Giardia isolates encompassing both human infecting G. duodenalis assemblages (A and B) and against metronidazole resistant parasites. These data suggest that SN00798527 has a different mode of action to metronidazole and that cross-resistance with the 5-nitroimidazoles is unlikely, and that the molecule is likely to be equally effective against both human infecting genotypes. Preliminary in vivo data suggests that this compound is well tolerated in Swiss mice, with no toxicity seen at oral doses of up to 0.7 mg/kg (10x the calculated therapeutic dose (CTD) for this compound). Preliminary data also suggest that SN00798527 is active in a murine giardiasis model. Neonate Swiss mice dosed daily for three days with orally administered SN00798527 (0.7 mg/kg 10xCTD) harboured significantly reduced parasite loads (73 % reduction in trophozoite load and 99 % reduction in cyst load) compared to untreated control mice. Taken together these data highlight the in vivo potential of series CL9596 and suggest further in vivo trials and mode of action studies are warranted. Lead compounds within two additional compound series (SC003542 and CL9406), SN00776497 and SN00797640 also demonstrated promising in vitro activity (48 h IC50 values 183 and 23 nM and SI of 291-343 and 24-90, respectively) that was consistent against multiple isolates encompassing both human infecting G. duodenalis genotypes and against metronidazole resistant parasites. While the timeframe of this project did not permit the in vivo anti-Giardia activity of these molecules to be evaluated, toxicity studies in Swiss mice also demonstrated these compounds to be safe at doses of up to 2 mg/kg and 5 mg/kg respectively (10x CTD).
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text

The concern of a Cryptosporidium or Giardia waterborne outbreak due to treated wastewater has had water treatment utilities using some of the highest water cleansing technologies available. Cryptosporidiosis and Giardiasis are severe diarrheal diseases which can lead to death, thus it is important that appropriate steps are taken to assure these parasites are not present in the effluent of treated wastewater. This study examined the results of 863 assays for Giardia and Cryptosporidium on the effluent of wastewater treatment facilities and found that county of collection, watershed of collection, and laboratory analyzing the sample have the most significant impact on the detection of Cryptosporidium oocysts and Giardia cysts in wastewater effluent and that there were minimal but significant differences in method of treatment and method of filtration. To date no other comprehensive analysis of this data has been done.

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FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Doenças tropicais negligenciadas (DTNs), assim classificadas pela Organização Mundial de Saúde, constituem um grupo de infecções praticamente eliminadas no mundo desenvolvido, mas que persistem em áreas pobres de países de baixa renda. Dentre elas estão a ascaridíase, a tricuríase e a ancilostomíase, cujos agentes etiológicos têm parte de seu ciclo evolutivo passando pelo solo, e são, por isso, denominadas de geohelmintoses. Recentemente, a giardíase também foi incluída como DTN, uma vez que sua forma de transmissão pode ser associada com falta de saneamento. Este estudo transversal de base populacional visou à investigação da ocorrência dessas parasitoses em três municípios do sudeste de Minas Gerais. Para tanto, 2367 indivíduos foram selecionados aleatoriamente. Os dados foram obtidos por meio de um questionário estruturado acerca das condições socioeconômicas, ambientais e culturais da população amostral e do exame de uma única amostra fecal de cada participante, pelo método de sedimentação espontânea (HPJ). Para cada amostra fecal foram examinadas cinco lâminas, e concluiu-se que o exame de três lâminas por amostra expressou a melhor relação custo-benefício para o SUS. A análise dos resultados mostrou que 6,1% (n=145) da população amostral estava infectada com uma ou duas DTNs. Dentre os indivíduos infectados, as prevalências observadas para ancilostomídeos, G. lamblia, T. trichiura e A. lumbricoides foram 47,3%, 27,3%, 16,0% e 9,3%, respectivamente. Na análise bivariada dos dados (p<0,05; IC 95%), o teste do Qui-quadrado de Pearson foi usado para avaliar a força das possíveis associações entre as variáveis independentes e o desfecho. Na análise de regressão logística, quatro variáveis permaneceram estatisticamente significantes, sugerindo serem os possíveis fatores de risco para as DTNs nos municípios: o destino inadequado do esgoto (p<0,001), o hábito de beber água não potável (p<0,001), a falta de instalação sanitária adequada (p=0,015) e pertencer ao sexo masculino (p<0,001). Os resultados encontrados neste estudo nos permitem concluir que DTNs estão presentes mesmo em regiões mais desenvolvidas do Brasil. O número de pessoas parasitadas confirma a área como de baixa endemicidade para geohelmintoses e giardíase em relação a outras regiões do Estado, e justifica estudos que avancem no conhecimento do perfil epidemiológico da região e subsidiem o desenvolvimento de políticas públicas locais.
Neglected tropical diseases (NTDs), so classified by the World Health Organization, are a group of infections that have been eliminated in the developed world, but still persist in poor areas of low-income countries. Among them, ascariasis, trichuriasis, and hookworm infection are soil-transmitted helminthiasis. Recently, and despite its worldwide distribution, giardiasis was also included in the WHO list, due to its way of transmission, which can be associated with lack of sanitation. This population-based cross-sectional survey aimed at investigating the occurrence of such infections in three municipalities of southeastern Minas Gerais state. A total of 2,367 individuals randomly chosen provided one single fecal sample. The baseline data consisted of a questionnaire and parasitological coproscopy using the HPJ method. Five slides were examined from each fecal sample, allowing us to conclude that, regarding this method, the examination of three slides per sample presented a satisfactory sensitivity and the best cost-effectiveness for the SUS. The results showed that 6.1% (n=145) of the population sample was infected with either one or two NTDs, of which the prevalences observed for hookworms, G. lamblia, T. trichiura, and A. lumbricoides were 47.3%, 27.3%, 16.0% e 9.3%, respectively. In the bivariate analysis (p<0.05; CI 95%), Pearson Qui-square tests were used to assess the strength of the associations. Multivariate analysis was run with all significant variables. Four variables remained significant in the logistic regression, as the possible risk factors for the installation and spread of the infections in the municipalities: inadequate sewage discharge (p<0.001), the habit of drinking unsafe water (p<0.001), lack of adequate sanitary infrastructure in the household (p=0.015), and masculine sex of the individual (p<0.001). Our findings allow us to conclude that NTDs occur even in more developed areas of Brazil. Our numbers confirm the study area as low endemic for giardiasis and soil-transmitted helminthiasis, and justify surveys in order to get more information on the epidemiological profile of the Zona da Mata Mineira region, aiming at giving support to the development of local health policies.

Лямбліоз входить в число найбільш поширених паразитарних захворювань в світі. За даними Всесвітньої організації охорони здоров'я, десята частина населення земної кулі інвазована лямбліями. Розрізняють три основні шляхи передачі лямбліозу: водний, контактно-побутовий і харчовий. Паразит викликає не тільки механічне подразнення стінки тонкого кишечнику, але і може призвести до патологічних рефлекторних реакцій з боку органів травлення.

O presente relatório descreve as principais atividades desenvolvidas no decorrer do estágio realizado em saúde pública e inspeção sanitária na Câmara Municipal de Manteigas. A área da sanidade animal mostrou-se a mais relevante em termos casuísticos. O protozoário do género Giardia spp., foi alvo de revisão bibliográfica neste trabalho, por constituir um problema de saúde pública devido ao seu potencial zoonótico. No decorrer do estágio, foi desenvolvido um estudo de prevalência de infeção por Giardia spp. em gatos domésticos e errantes, constituindo o concelho de Manteigas a área geográfica alvo de pesquisa. Foram recolhidas 34 amostras fecais para pesquisa de quistos de Giardia pelo método de flutuação fecal, seguido de um teste imunocromatográfico para deteção de antigénios de Giardia para confirmação diagnóstica. Verificou-se uma prevalência de 11,8% de resultados positivos para Giardia spp. Em felinos do concelho, correspondentes a 4 animais positivos dos 34 testados; ABSTRACT: Internship report on public health and sanitary inspection and study the prevalence of Giardia spp. Infection in cats in the municipality of Manteigas This report describes the main activities performed during the internship in public health and sanitary inspection at Manteigas City Hall. The field of animal health proved to be the most relevant terms on case. The protozoan of the genus Giardia spp., was the target of a literature review in this work, since it’s a problem of public health because of the zoonotic potential. During the internship the study on the prevalence of Giardia spp. infection was developed in both domestic and stray cats, the geographical target area of research was the civil parish of Manteigas. Were collected 34 fecal samples, for the detection of Giardia cysts by fecal flotation method, followed by an immunoassay for detection of Giardia antigen to confirm the diagnosis. There was 11, 8% prevalence of this parasite in cats in the civil parish, referring to 4 of the 34 animals tested positive.

Agriculture is key to New Zealand�s economy with land-use conversions in response to market forces occurring regularly. Recently, high-intensity dairy farming has replaced low-density livestock farming, often degrading surrounding waterways. Of particular concern is that dairy cattle can be a source of the parasite Giardia, which in humans is a common cause of gastrointestinal infection. Thus, this thesis evaluated whether dairy farm conversions posed significant consequences for public health. First I examined the prevalence of Giardia in calves in a rapidly intensifying dairying region of New Zealand. A total of 1190 faecal samples were collected from calves one to seven weeks old during two spring calving seasons and screened by direct immunofluorescent microscopy. Giardia cysts were detected in 31% of samples. To evaluate the potential risk that this environmental source of Giardia posed to the human population, molecular genotyping was used to compare forty Giardia strains isolated from calves with thirty isolates from humans collected in the same region and period. Sequencing the β-giardin gene, Giardia duodenalis assemblages A and B were identified from both hosts, with genotype comparisons revealing substantial overlap of identical genotypes for both assemblages, implying zoonotic transmission. Environmental agencies routinely promote the planting of streamside edges to decrease nonpoint pollution from dairy farms entering waterways. However, current methods for tracking pathogens across farmland and into waterways via surface runoff are limited and typically have been developed using artificially created landscapes. Furthermore, no studies have investigated how Giardia moves across the landscape in farm surface runoff. I developed a field-based tracking method specific for Giardia and used this technique to compare the ability of recently planted vegetation strips with bare soil strips cleared of vegetation at decreasing pathogen concentrations; a typical scenario when planting barriers to reduce waterway contamination. A spike containing a bromide tracer and inactivated Giardia cysts was applied in drip-irrigated surface runoff, with one-minute samples collected from the bottom of the plot. A significant treatment effect was identified for Giardia, with 26% fewer detected in runoff from the planted strip, highlighting the immediate benefit of vegetation planting in removing pathogens. Next I evaluated the effects of four riparian treatments on Giardia runoff: exotic pasture grass and weeds growing in the absence of cattle grazing due to fencing, in comparison to monocultural plantings of three New Zealand native grassland species. Runoff experiments were performed after planting, both prior to and following the main summer growing season. Bromide recovery was high from all four treatments (54 - 99%), with no significant treatment effects. By comparison, Giardia recovery was low (1 - 13%). Prior to summer, two native species reduced Giardia in runoff more than the pasture grass/weed treatment which was almost vegetation-free at this time. After summer, Giardia recoveries were uniformly lower in all treatments. These results demonstrate that after one growing season, fencing waterways produces riparian buffers, via the growth of exotic pasture plants released from grazing, that decrease pathogen concentrations in surface runoff to concentrations indistinguishable from native plantings. Given infectious organisms are known to be in the environment, it is important to assess the risk these pose to human populations. Findings from this research can be used to improve currently available risk-assessment models for Giardia transmission from infected dairy animals via water to humans.

Orientadores: Regina Maura Bueno Franco, Eliete Maria Silva, Jancarlo Ferreira Gomes
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Parasitoses intestinais são frequentes na população usuária do Sistema Único de Saúde de São Sebastião da Grama, município do interior do estado de São Paulo. Durante a ocorrência de um surto de gastroenterite em uma das creches do município, objetivou-se investigar a ocorrência de enteroparasitos nos trabalhadores e crianças das instituições municipais urbanas de educação infantil, avaliar o desempenho de técnicas diagnósticas na detecção de Giardia duodenalis em amostras fecais provenientes de creche, local de surto de gastroenterite, e implantar um programa de controle desta parasitose, o "Programa de Controle da Giardiose em Creches e Pré-escolas" (PCGCP) a fim de avaliar a adesão de gestores, trabalhadores e famílias das crianças às medidas higiênicas no ambiente escolar infantil. Para tanto se utilizou amostras fecais de 172 crianças e 33 trabalhadores, processadas pelos métodos Three Fecal Test Conventional® e Modified®, diagnosticando-se 89,53 % das crianças e 71,87 % dos trabalhadores positivos para parasitos e comensais. Blastocystis hominis foi o parasito intestinal de maior prevalência entre as crianças (86,62 %) e trabalhadores (65,62 %). Os casos de monoparasitismo foram mais frequentes que os de poliparasitismo entre as crianças e os trabalhadores estudados, bem como a prevalência de protozoários foi maior que a de helmintos. O modelo de regressão logística evidenciou associação significativa entre criança atendida na creche e a frequência de infecção por enteroparasitos (p = 0,01), por G. duodenalis (p = 0,00), por B. hominis (p = 0,02), e pelos protozoários intestinais (p = 0,01). Entre criança menor de um ano e a frequência de infecção por G. duodenalis (p = 0,00), e entre crianças cujos domicílios não possuíam coleta de lixo e a frequência de infecção por helmintos (p = 0,03). Os resultados obtidos utilizando-se os métodos de Faust et al. e ELISA foram concordantes substancialmente, contudo, a presença de resultados falsos positivos verificados no imunoensaio limitam sua aplicabilidade como teste diagnóstico em uma situação de surto. Como ferramenta de controle e prevenção da giardiose, foi implantado e avaliado o PCGCP nestes ambientes do estudo. A adesão e ativo engajamento dos gestores, trabalhadores e famílias das crianças ao PCGCP foi considerada satisfatória, e os resultados sugerem que o treinamento das práticas para se evitar a giardiose deva ser mantido de forma permanente a fim de se alcançar uma efetiva prevenção nos ambientes escolares infantis
Abstract: Intestinal parasites are common in the population using the Unified Health System of São Sebastião da Grama, a municipality in the state of São Paulo. The aims of this study to investigate the occurrence of intestinal parasites in children and workers of urban municipal institutions of early childhood education, to evaluate the performance of diagnostic techniques for detection of Giardia duodenalis in stool samples from daycare centers, where outbreaks of gastroenteritis occur. To implement a program to control this parasite, the "Program of Control of Giardiasis in Day Care Centers and Preschools" (PCGCP), in order to assess the commitment of managers, workers and children?s families to hygienic measures within these school environments. For this purpose we used fecal samples from 172 children and 33 workers, processed using the Conventional and Modified Three Fecal Test® method. As a result, 89,53 % of the children and 71,87 % of workers were diagnosed positive for pathogenic parasites and commensals. Blastocystis hominis was the most prevalent intestinal parasite among children (86,62 %) and employees (65,62 %). Monoparasitism cases were more frequent than those of multiple parasitic infections among children and workers. The prevalence of protozoa was greater than that of helminths. The logistic regression model revealed a significant association between child attended in the day care center and the frequency of infection with intestinal parasites (p = 0,01), by G. duodenalis (p = 0,00), by B. hominis (p = 0,02), and the intestinal protozoa (p = 0,01). Higher indices were found among children under one year of age (frequency of infection with G. duodenalis (p = 0,00)) and among children whose homes had no waste collection (frequency of helminth infection (p = 0,03)).The results obtained using the methods of Faust et al. and ELISA were substantially consistent. However, the presence of false positive results observed in the immunoassay limits its applicability as a diagnostic test in an outbreak situation. As a tool for control and prevention of giardiasis, the program PCGCP was implemented and its adherence was evaluated in the sites investigated, places considered at the scope of this study. The commitment and active engagement of managers, workers and families with children to PCGCP was considered satisfactory, and the results suggest that training practices to avoid giardiasis should be made permanent for an effective prevention in day care centers and preschools environments to be achieved
Mestrado
Parasitologia
Mestre em Parasitologia

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Foz do Iguaçu has today more than 81 ethnicities, associated to a wide cultural diversity they contribute to the divergence of hygienic habits, resulting in precarious sanitary conditions. Fir this reason, the city government has been presenting several changes in public health area. The city is also located on a tri-border border area, about 7 km from Ciudad Del Este (Paraguay) and 10 km from Puerto Iguazu (Argentina). Its main economic product is Tourism, due to the relevance of the Iguazu Falls, considered one of the seven wonders of nature and Itaipu Hydroelectric Power Plant, which is on the border of Brazil and Paraguay. Because of the construction of the Hydroelectric Power Plant, in just 20 years, Foz do Iguaçu has grown by 383% in the total population of the city, generating a great job offer. Due to the increasing number of women entering the labor market, the Municipal Early Childhood Centers (CMEIs) have been a reality in the life of families, as this is the place where many children spend most of their time while mothers are at work. These institutions play an important role in the development of children as a whole, mainly referring to the intellectual, physical, social and psychological aspects. Children attending CMEIs are more susceptible to infections than those who are kept in their homes, which can be explained by the large interpersonal contact provided by the collective environments. To verify the prevalence of parasitic diseases among children attending CMEIs in Foz do Iguaçu and to analyze the possible relationship with the socioeconomic, environmental and hygienic scenario of children, parents and caregivers. This is a field research, with descriptive and quantitative approaches. The studied population consisted of all pre-school children aged from 3 to 5 years old, enrolled in CMEIs A and B, and parents and/or guardians working with sampling by convenience. The feces of the children were collected and then sent for analysis in the Environmental Laboratory of the Itaipu Hydroelectric Power Plant. In addition, a questionnaire was applied to the parents or guardians of the children, containing 19 closed questions, regarding the factors that contribute to the merge of parasitic diseases. Data were analyzed by the BioEstat 5.3® program and, the level of significance was considered at 5%. Results: A total of 82 fecal samples were analyzed from February to June 2016. Positive results for parasites were found in 38.8% of them. The following etiological agents were found: Giardia duodenalis (28,0%), Hymenolepis nana (3,6%), Ascaris lumbricoides (2,4%), Entamoeba coli (2,4%) and Enterobius vermicularis (2,4%). Regarding the degree of parasitism, 35.0% of the children presented monoparasitism and only 4% presented biparasitism, and there were no cases of polyparasitism. The parents and guardians of the parasitized children (32/82) received the antiparasitic medication prescription (metronidazole) and the medicament for the treatment of the children. In addition, they received guidance in order to prevent the emergence of new cases. The most affected gender by parasites was the male (20/41), followed by the female (12/41), (p<0.05) in the age range of 3 to 4 years (84.3%). The socioeconomic profile revealed that the level of schooling of mothers, complete secondary education (15/29 - p<0.05), and incomplete high school parents (13/24 - p>0.05), family income up to one Minimum wage (19/47 - p<0.05), residential structure with piped water and treated with chlorine (32/80 - p<0.05), waste disposal by means of cesspool (12/36 - p<0.05), may have influenced the average incidence of parasitism, as well as the aspects related to the habit of washing hands after diaper change (21/47 - p<0.05) and the health of the child, the taking of feces exams (19/45), abdominal pain (25/65), nausea (31/79), vomiting (31/78), weight loss (28/71) and presence of formed faeces (17/43) ) (P<0.05). However, the prevalence of parasitoses was related to the form of cleaning foods (fruits, and vegetables), only with water (23/60 - p<0.05), with children who do not have the habit of washing their hands with soap before meals and after using the toilet (6/15 - p>0.05). Conclusion: The results indicate an important prevalence of intestinal parasitoses in children who live on the border area, in the age range of 3 to 5 years old, mainly giardiasis. The presence of parasitic diseases in this population provides an epidemiological profile that can help in the construction of public health policies which are more appropriate to this social reality, in order to contribute to the improvement of health education of the population, especially the family members responsible for the children who are the most affected by the to intestinal parasites. Keywords: parasitic
Foz do Iguaçu presenta 81 etnias, con una amplia diversidad cultural, contribuyendo a la divergencia de hábitos higiénicos, resultando en precarias condiciones sanitarias. Como consecuencia, el municipio viene presentando diversas transformaciones en el área de la salud. Además, se encuentra en la triple frontera, aproximadamente siete kilómetros de Ciudad Del Este (Paraguay) y a 10 km de Puerto Iguazu (Argentina). Su economía se basa principalmente en el turismo, debido a las Cataratas del Iguazú, la cual es considerada como una de las siete maravillas de la naturaleza y la Usina Hidroeléctrica de Itaipú. En los últimos 20 años, debido a la construcción de la Usina Hidroeléctrica Itaipú, el municipio de Foz do Iguaçu tuvo un crecimiento poblacional del 383%, convirtiéndose además en unas de las principales fuente de empleo. En razón del número cada vez mayor de mujeres insertas en el mercado de trabajo, los Centros Municipales de Educación Infantil (CMEIs) han sido una realidad en la vida de las familias, por ser este el lugar donde muchos niños pasan la mayor parte de su tiempo. Estas instituciones desempeñan un papel importantísimo en el desarrollo integral de los niños, principalmente relacionados con los aspectos intelectual, físico, social y psicológico. Los niños que frecuentan los CMEIs son más susceptibles a las infecciones que las que se mantienen en sus hogares, lo que puede ser explicado por el gran contacto interpersonal propiciado por los ambientes colectivos. Verificar la prevalencia de parasitosis en niños matriculadas en los CMEIs de Foz do Iguaçu y analizar la posible relación con el escenario socioeconómico, ambiental e higiénico de los niños, de los padres y/o responsables. Materiales y Métodos: Se trata de una investigación de campo, descriptiva y de abordaje cuantitativo. La población del estudio se constituyó de todos los niños con edad preescolar comprendida entre los 3 y 5 años, matriculados en los CMEIs A y B, y de los padres y / o responsables donde se trabajó con el muestreo de conveniencia. Se realizó la recolección de examen parasitológico de heces en los niños matriculados en los CMEIs y posteriormente procesados en el Laboratorio Ambiental de la Usina Hidroeléctrica de Itaipú. Además, se aplicó un cuestionario a los padres o responsables de los niños, conteniendo 19 preguntas cerradas, referentes a los factores contribuyentes para el surgimiento de enfermedades parasitarias. Los datos fueron analizados por el programa BioEstat 5.3® y, considerado el nivel de significancia en el 5%. Se analizaron 82 muestras fecales en el período de febrero a junio de 2016, obteniéndose positividad para parásitos en (38,8%), siendo encontrados los siguientes agentes etiológicos: Giardia duodenalis (28,0%), Hymenolepis nana (3,6%), Ascaris lumbricoides (2,4%), Entamoeba coli (2,4%) y Enterobius vermicular (2,4%). En cuanto al grado de parasitismo, el 35,0% de los niños presentaron monoparasitismo y sólo el 4% presentó biparasitismo, no habiendo casos de poliparasitismo. Los padres y/o los responsables de los niños parásitos (32/82), recibieron la prescripción medicamentosa de antiparasitario (metronidazol) y el medicamento para el tratamiento de los niños. Además, recibieron orientaciones para prevenir el surgimiento de nuevos casos. El género, más propicio por parasitosis fue el masculino (20/41), seguido del femenino (12/41), (P<0,05) con rango de edad de 3 a 4 años (84,3%). El perfil socioeconómico reveló que el grado de escolaridad de las madres, enseñanza media completa (15/29 - p<0,05), y de los padres de secundaria incompleta (13/24 - p>0,05), ingreso familiar de hasta un año Salario mínimo (19/47 - p<0,05), estructura residencial con agua potable y tratada con cloro (32/80 - p<0,05), eliminación de los desechos por medio de fosa (12/36 - p<0,5), puede haber influido en la incidencia media de parasitismo, así como los aspectos relacionados con la higienización de las manos de los responsables de los niños después del cambio de pañales (21/47 - p<0,05) y de la salud del niño, realización De examen parasitológico de heces (19/45), dolor abdominal (25/65), náuseas (31/79), vómitos (31/78), pérdida de peso (28/71) y presencia de heces formadas (17/43) (P<0,05). Sin embargo, la prevalencia de parasitosis fue relacionada con la forma de higienización de alimentos (frutas, verduras y legumbres), solamente con agua (23/60 - p<0,05), con los niños que no tienen el hábito de lavarse las manos Con uso de jabón antes de las comidas y después del uso del sanitario (6/15 - p>0,05). Conclusión: Los resultados apuntan a una prevalencia importante de parasitosis intestinal en niños de la región de frontera con edad comprendida entre los 3 a 5 años, principalmente de giardiasis. La presencia de enfermedades parasitarias en esta población proporciona un perfil epidemiológico que puede ayudar en la construcción de políticas públicas de salud más adecuadas a esa realidad social, para contribuir a mejorar la educación en salud de la población, en especial a los familiares responsables por los niños afectados por parasitosis intestinales.
Foz do Iguaçu apresenta mais de 81 etnias, associadas a uma ampla diversidade cultural, contribuindo para a divergência de hábitos higiênicos, resultando em precárias condições sanitárias. Como decorrência, o município vem apresentando diversas transformações na área da saúde. Além disso, encontra-se na tríplice fronteira, a cerca de sete km de Ciudad Del Este (Paraguai) e 10 km de Puerto Iguazu (Argentina). Tem como principal base de economia o turismo, devido a Cataratas do Iguaçu, considerada umas das sete maravilhas da natureza e a Usina Hidrelétrica de Itaipu, que fica na divisa do Brasil com o Paraguai. Devido a construção da Usina Hidrelétrica, em apenas 20 anos, Foz do Iguaçu apresentou um crescimento de 383% de habitantes no total da população do município, gerando grande oferta de emprego. Em razão do número cada vez maior de mulheres inseridas no mercado de trabalho, os Centros Municipais de Educação Infantil (CMEIs) tem sido uma realidade na vida das famílias, por ser este o local onde muitas crianças passam a maior parte do seu tempo. Estas instituições têm um papel importantíssimo no desenvolvimento integral das crianças, principalmente relacionados aos aspectos intelectual, físico, social e psicológico. As crianças que frequentam os CMEIs são mais suscetíveis às infecções do que aquelas que são mantidas em suas residências, o que pode ser explicado pelo grande contato interpessoal propiciado pelos ambientes coletivos. Objetivou-se verificar a prevalência de parasitoses em crianças atendidas em CMEIs de Foz do Iguaçu e analisar a possível relação com o cenário socioeconômico, sanitário e higiênico das crianças, dos pais e ou responsáveis. Trata-se de uma pesquisa de campo, descritiva e de abordagem quantitativa. A população do estudo foi constituída de todas as crianças com idade pré-escolar de 3 a 5 anos, matriculadas nos CMEIs A e B, e dos pais e ou responsáveis onde se trabalhou com amostragem de conveniência. Foi realizada a coleta de exame parasitológico de fezes nas crianças matriculadas nos CMEIs e, posteriormente processadas no Laboratório Ambiental da Usina Hidrelétrica de Itaipu. Além disso, foi aplicado um questionário aos pais ou responsáveis das crianças, contendo 19 questões perguntas fechadas, referentes aos fatores contribuintes para o surgimento de doenças parasitárias. Os dados foram analisados pelo programa BioEstat 5.3® e, considerado o nível de significância em 5%. Foram analisadas 82 amostras fecais no período de fevereiro a junho de 2016, obtendo-se positividade para parasitos em 38,8%, sendo encontrados os seguintes agentes etiológicos: Giardia duodenalis (28,0%), Hymenolepis nana (3,6%), Ascaris lumbricoides (2,4%), Entamoeba coli (2,4%) e Enterobius vermicularis (2,4%). Em relação ao grau de parasitismos, 35,0% das crianças apresentaram monoparasitismo e apenas 4% apresentaram biparasitismo, não havendo casos de poliparasitismo. Os pais e ou responsáveis das crianças parasitadas (32/82), receberam a prescrição medicamentosa de antiparasitário (metronidazol) e o medicamento para o tratamento das crianças. Além disso, receberam orientações no intuito de se prevenir o surgimento de novos casos. O gênero, mais acometido por parasitoses foi o masculino (20/41), seguido do feminino (12/41), (p<0,05) com faixa etária de 3 a 4 anos (84,3%). O perfil socioeconômico revelou que o grau de escolaridade das mães, ensino médio completo (15/29 - p<0,05), e dos pais ensino médio incompleto (13/24 - p>0,05), renda familiar de até um salário mínimo (19/47 – p<0,05), estrutura residencial com água encanada e tratada com cloro (32/80 – p<0,05), eliminação dos dejetos por meio de fossa (12/36 - p<0,05), pode ter influenciado para a incidência média de parasitismo, assim como os aspectos relacionados a higienização das mãos dos responsáveis das crianças após a troca de fraldas (21/47 - p<0,05) e da saúde da criança, realização de exame parasitológico de fezes (19/45), dor abdominal (25/65), náuseas (31/79), vômitos (31/78), perda de peso (28/71) e presença de fezes formadas (17/43) (p<0,05). No entanto, a prevalência de parasitoses foi relacionada à forma de higienização de alimentos (frutas, verduras e legumes), somente com água (23/60 - p<0,05), com as crianças que não tem o hábito de lavar as mãos com uso de sabão antes das refeições e após o uso do sanitário (6/15 – p>0,05). Os resultados apontam uma prevalência importante de parasitoses intestinais em crianças fronteiriças na faixa etária de 3 a 5 anos, principalmente de giardíase. A presença de doenças parasitárias nesta população fornece um perfil epidemiológico que pode auxiliar na construção de políticas públicas de saúde mais adequadas a essa realidade social, de forma a contribuir na melhoria da educação em saúde da população, em especial dos familiares responsáveis das crianças atingidas pelas parasitoses intestinais.

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Com o objetivo de avaliar a influência que a infecção por parasitas intestinais exerce sobre o estado antropométrico na infância, na cidade de Salvador-BA, foi desenvolvido um estudo seccional em uma amostra probabilística de 629 crianças entre 12 e 48 meses. Aplicou-se a técnica de regressão logística multivariada, tendo como variáveis dependentes os indicadores antropométricos peso por idade (P/I) e altura/comprimento por idade (A/I). A presença ou ausência de diferentes parasitas nas fezes (A. lumbricoides, T. trichiura, G. duodenalis), constituiu-se na variável independente. O parasita mais freqüente foi A. lumbricoides (23,1%), seguido de T. trichiura (16,5%) e G. duodenalis (13,5%). Entre as crianças infectadas a média de z-scores dos indicadores antropométricos foi menor do que a observada entre as crianças não infectadas. Dentre as infecções estudadas, apenas a giardíase esteve significativamente associada ao déficit de crescimento linear, incluindo quando a freqüência da coleta pública de lixo era inadequada (modificador de efeito). Observam-se associações com o déficit antropométrico, segundo o indicador P/I, na presença da giardíase quando a criança não era amamentada por mais de seis meses (OR=2,92; IC95%=1,62-5,24) e quando residia em domicílio cuja pavimentação da rua/calçada era inadequada (OR=3,04; IC95%=1,53-6,07), após ajuste por confundidores. Os resultados deste estudo evidenciaram que, em crianças com giardíase, a amamentação durante o período recomendado pela OMS constituiu-se em um fator de proteção para o estado antropométrico e que o efeito negativo das condições ambientais indesejadas referentes ao lixo urbano e à pavimentação sobre o estado antropométrico pode ser devido à provável mais elevada carga parasitária existente nas crianças infectadas por este protozoário, vivendo neste contexto insalubre.
Salvador

Giardia duodenalis est le protozoaire responsable de la giardiose, la parasitose intestinale la plus répandue dans le monde. Cette infection se caractérise par une malabsorption intestinale, des diarrhées, une perte de poids et des douleurs abdominales intenses chez l’Homme et de nombreux mammifères. Par ailleurs, cette maladie dont l’impact en santé publique et vétérinaire est reconnu, peut entraîner d’importantes déficiences nutritionnelles en particulier chez les sujets jeunes. L’infection est causée par l’ingestion de kystes de Giardia duodenalis (syn. G. lamblia, G. intestinalis) présents dans les aliments ou l’eau contaminée. Infectieux à très faibles doses, ces kystes survivent pendant plusieurs semaines dans l’environnement et sont résistants aux différents désinfectants. Suite au dékystement, la forme végétative de Giardia, le trophozoïte, adhère à l’épithélium intestinal au niveau des parties supérieures de l’intestin grêle et se multiplie, causant les symptômes. Cette phase se termine par un nouvel enkystement et l’excrétion de kystes par les fèces. Le nombre croissant d’infections liées à la contamination de l’eau potable, à l’émergence de souches résistantes aux médicaments disponibles, à la fréquence des échecs thérapeutiques et à l’importance des effets secondaires associés aux traitements font de cette maladie un sujet d’actualité de plus en plus préoccupant qui nécessite le développement de traitements alternatifs. Il est désormais bien établi que le microbiote et/ou certaines souches de bactéries probiotiques ont un impact bénéfique dans la giardiose. En particulier, la bactérie probiotique Lactobacillus johnsonii La1 (LjLa1) a un rôle protecteur contre la croissance de Giardia in vitro et in vivo. Nous avons cherché dans ce travail de Thèse à décrypter les mécanismes moléculaires associés à l’effet inhibiteur des facteurs sécrétés par LjLa1. Nous avons montré qu’in vitro, LjLa1 agissait en libérant des enzymes de type Bile Salt Hydrolases (BSH) qui modifient alors des composants de la bile non-toxiques pour le parasite (sels biliaires conjugués) en des composants toxiques (sels biliaires déconjugués). Les 3 gènes BSH codés dans le génome de LjLa1 ont été clonés chez Escherichia coli et les protéines taguées histidine purifiées pour étudier leurs propriétés biochimiques et biologiques. Obtenues sous forme active, nous avons pu en définir les spécificités de substrats et montrer qu’elles sont capables d’inhiber significativement la croissance de G. duodenalis in vitro et in vivo, dans un modèle murin de la giardiose (souriceaux OF1 non sevrés). En parallèle, nous avons identifié, à l’issue d’un large criblage de souches de lactobacilles selon leur activité anti-Giardia in vitro, une souche probiotique aux effets inhibiteurs comparables à ceux de LjLa1 : Lactobacillus gasseri CNCM-4884. Administrée in vivo dans le modèle murin de la giardiose, cette souche a réduit de 93% la charge parasitaire dans l'intestin grêle des nouveaux nés et a également réduit de façon significative le nombre de kystes libérés dans l’environnement, permettant ainsi de réduire la transmission de Giardia. Des travaux parallèles ont été réalisés au cours de ce projet de Thèse, notamment le développement d’outils de moléculaire pour l’expression hétérologue de molécules d’intérêt en santé animale chez divers lactobacilles. Le développement de ces « vecteurs mucosaux » permettra à terme de proposer une stratégie de surexpression de BSH par les lactobacilles afin d’accroitre l’activité BSH in vivo, et renforcer ainsi l’élimination du parasite. Ces résultats permettent de proposer de nouvelles pistes thérapeutiques originales contre les giardioses humaines et animales, basées sur l’utilisation de lactobacilles probiotiques ou sur les activités BSH qui en sont dérivées. Ces traitements offrent alors une alternative sérieuse aux antibiotiques et permettront de pallier aux actuels fréquents échecs thérapeutiques
Giardia duodenalis is a protozoan parasite responsible for giardiasis, the most common intestinal parasitic disease worldwide. This infection is characterized by intestinal malabsorption, diarrhea, weight loss and abdominal pain in humans and various mammalian species. Besides, this disease has a high veterinary and public health impact, leading to important nutritional deficiencies in young subjects. The infection is caused by the ingestion of food or water contaminated with infectious cysts of the parasite. Giardia cysts can survive for several weeks in the environment and are highly resistant to disinfectants. Giardia excysts in the intestinal tracts of its host and replicates under the trophozoite stage causing the symptoms. Trophozoites adhere to the intestinal epithelium of the small intestine and multiply, causing the symptoms. The cycle ends by a new encystment and infectious cysts are released in environments with feces. The increasing number of giardiasis cases, mainly due to water contaminations, the emergence of parasite strains resistant to drugs and therapeutic failures, make research on alternative therapeutic strategies and treatments highly needed. Nowadays, it is well known that the microbiota and probiotics play an important role in protection against this parasite. For instance, the probiotic strain Lactobacillus johnsonii La1 (LjLa1) prevents the establishment of Giardia in vitro and in vivo. In this thesis, we have tried to point out the molecular mechanism(s) involved in this inhibitory effect(s). We showed in vitro that LjLa1 was releasing "Bile Salt Hydrolases" (BSH) – like activities that modify some components of bile (conjugated bile salts) into toxic compounds (deconjugated bile salts) for Giardia. We have cloned and expressed each of the three bsh genes present in the genome of LjLa1 in Escherichia coli in order to study their enzymatic and biological properties. Two BSH were obtained as recombinant active enzymes and biochemical tests showed that they have distinct substrate specificities despite similar predicted 3D structures. Moreover, these two BSHs of LjLa1 exhibited anti-giardial effects in vitro and in vivo in a murine model of the giardiasis (OF1suckling mice), comforting the hypothesis of the biological role of active BSH, derived from probiotics, against Giardia. A wide collection of diverse lactobacilli strains was screened to assess their effectiveness to also display both anti-giardial and BSH activities. This screening allowed the identification of several strains exhibiting strong anti-giardial effects such as Lactobacillus gasseri CNCM I-4884. In a murine model of giardiasis, this strain dramatically reduced the parasite burden in the small intestine of treated animals and significantly reduced the number of cysts in the colon, which could contribute to blockage of parasite transmission in environments. Additional studies were realized in parallel in order to explore the potency of lactobacilli to exert beneficial effects on health. For this, molecular tools were successfully developed in various lactobacilli strains to express and deliver therapeutic molecules at mucosal surfaces. The development of these tools will further allow the overexpression of BSH by lactobacilli to increase their in vivo BSH-activity and strengthen the elimination of the parasite. Altogether, this thesis work proposes new original therapeutic strategies against human and animal giardiasis, based on the use of BSH-positive lactobacilli strains or recombinant BSH- derived from probiotic strains, to counteract the frequent therapeutic failures, offering a serious alternative to antibiotics

A giardíase é uma doença entérica de alta prevalência particularmente nos países em desenvolvimento. Diferentes espécies foram descritas em função de seus hospedeiros. A G. duodenalis é a espécie que parasito não só o homem, mas também animais domésticos e silvestres. Recentemente, com a aplicação de técnicas moleculares, foi possível identificar sete diferentes agrupamentos sendo que cada um deles tem predileção por determinadas espécies de hospedeiro. Essa discriminação não é possível por meio da microscopia. A identificação do(s) agrupamento(s) presente(s) na população de determinada região pode ser de grande importância na elaboração de políticas de saúde pública já que revela o perfil de transmissão desse protozoário. Este estudo teve por objetivos verificar o grau de positividade de enteroparasitos, a contribuição de cada um deles nesse índice e o genótipo dos cistos de G. duodenalis presentes em amostras de fezes de indivíduos atendidos no Hospital Universitário da Universidade de São Paulo. Foram analisadas 6717 amostras com positividade para algum enteroparasito de 12,5%, sendo S.stercoralis e G. duodenais respectivamente o helminto e protozoário patogênico mais frequentes. A maioria dos casos de G. duodenalis genotipada era dos agrupamentos AII e B.
Giardiasis is a very prevalent enteric disease occurring mainly in developing countries. Different species have been described concerning their hosts. G. duodenalis parasites not only men but also domestic and wild animals. Recently molecular techniques have been used to identify seven different assemblages which parasites specific hosts. The microscopic analysis do not allows that discrimination. It can be useful to know the assemblage profile of specific population in order to provide some important information to built public health politicies once it reveals the transmition of the protozoan The aim of this study was to verify the enteroparasites infection degree, the individual contribution of each one in this picture and to genotyping G. duodenalis cysts from human faecal samples of individuals from University Hospital from Universty of São Paulo. So, 6717 faecal samples were analysed and 12,5% were positive for some enteroparasite and S.stercoralis and G. duodenais were the helminth e pathogenic protozoan more frequently found. Most of the cases of G. duodenalis were from the assemblages AII and B.