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Universidade do Estado do Par?. Programa de P?s-Gradua??o Strictu Sensu em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brasil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Parasitoses Intestinais, Esquistossomose e Malacologia. Ananindeua, PA, Brasil. Universidade do Estado do Par?. Programa de P?s-Gradua??o Strictu Sensu em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brasil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Parasitoses Intestinais, Esquistossomose e Malacologia. Ananindeua, PA, Brasil. Universidade do Estado do Par?. Programa de P?s-Gradua??o Strictu Sensu em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brasil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Parasitoses Intestinais, Esquistossomose e Malacologia. Ananindeua, PA, Brasil. Universidade do Estado do Par?. Programa de P?s-Gradua??o Strictu Sensu em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brasil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Parasitoses Intestinais, Esquistossomose e Malacologia. Ananindeua, PA, Brasil. Pontif?cia Universidade Cat?lica do Rio Grande do Sul. Programa de P?s-Gradua??o em Medicina e Ci?ncias da Sa?de. Laborat?rio de Parasitologia Biom?dica. Porto Alegre, RS, Brasil. Universidade Federal de Minas Gerais. Departamento de Parasitologia. Belo Horizonte, MG, Brasil. Universidade do Estado do Par?. Programa de P?s-Gradua??o Strictu Sensu em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brasil / Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Parasitoses Intestinais, Esquistossomose e Malacologia. Ananindeua, PA, Brasil. INTRODUCTION: Schistosomiasis is a poverty-related disease that affects people in 78 countries worldwide. This study aimed to evaluate the point-of-care circulating cathodic antigen (POC-CCA) test performance using sensitive parasitological methods as a reference standard (RS) in individuals before and after treatment. METHODS: The RS was established by combining the results of 16 Kato-Katz slides and the Helmintex? method. Positivity rates of the POC-CCA test and Kato-Katz and Helmintex? methods were calculated before treatment and 30 days afterward. Furthermore, the sensitivity, specificity, accuracy, and kappa coefficient before treatment were determined by comparing the methods. The cure rate was defined 30 days after treatment. RESULTS: Among the 217 participants, the RS detected a total of 63 (29.0%) positive individuals. The POC-CCA test identified 79 (36.4%) infections. The evaluation of POC-CCA test performance in relation to the RS revealed a sensitivity of 61.9%, specificity of 74.0%, accuracy of 70.5%, and kappa coefficient of 0.33. Out of the 53 remaining participants after treatment, a total of 45 (81.1%) showed egg negative results, and 8 (18.9%) were egg positive according to the RS. A total of 5 (9.4%) egg-positive and 37 (69.8%) egg-negative individuals were positive by the POC-CCA test. CONCLUSIONS: Our data show that the POC-CCA test has potential as an auxiliary tool for the diagnosis of Schistosoma mansoni infection, yielding better results than 16 Kato-Katz slides from three different stool samples. However, the immunochromatographic test lacks sufficient specificity and sensitivity for verifying the cure rate after treatment.

A esquistossomose humana ? apontada como uma das doen?as tropicais mais devastadoras, afetando cerca de 250 milh?es de indiv?duos em todo o mundo. Por este motivo, ? considerada a segunda doen?a parasit?ria mais importante em termos de efeito socioecon?mico, perdendo apenas para mal?ria. No Brasil, o Schistosoma mansoni ? o ?nico agente etiol?gico da esquistossomose e atinge 19 Estados. Em ?reas de recente introdu??o da doen?a ou onde ocorre o controle, a baixa carga parasit?ria ? uma realidade, e sua detec??o ? dificultada pela baixa sensibilidade diagn?stica dos m?todos dispon?veis. O Helmintex? ? um m?todo proposto recentemente, que utiliza uma grande quantidade de fezes, in?meros processos de concentra??o, e adi??o de part?culas paramagn?ticas, provando-se com alta sensibilidade para a detec??o de ovos de S. mansoni nas fezes. No entanto, ? um processo laborioso, com limitada aplica??o em campo e demora na entrega do diagn?stico. O foco principal desta Tese ? o aprimoramento de metodologias de detec??o de ovos para o diagn?stico da esquistossomose, buscando, principalmente, o melhoramento da metodologia Helmintex?. Desta forma, o primeiro cap?tulo desta Tese prop?e a redu??o do sedimento final Helmintex?, com o uso de detergente neutro, Tween 20, e a colora??o dos ovos de S. mansoni com ninidrina, buscando otimizar a metodologia. O segundo cap?tulo visa o uso de uma rea??o quimioluminescente para a detec??o de ovos S. mansoni em meio ao sedimento final Helmintex?, objetivando a rapidez no diagn?stico final. Por fim, o terceiro cap?tulo, explora o entendimento das peculiaridades dos ovos na sua passagem pela parede intestinal, observando a passagem em ?cluster? e secre??o, pelos ovos, de uma prote?na denominada Sm 16. Os resultados obtidos neste trabalho refor?am o papel proposto ao m?todo Helmintex, de servir como refer?ncia para o diagn?stico da esquistossomose intestinal, e para a avalia??o comparativa de outros testes, al?m de registrar pela primeira vez, a forma??o de ?cluster? e a excre??o da prote?na Sm16 por ovos de S. mansoni durante sua migra??o pela parede intestinal de camundongos. Human schistosomiasis is considered one of the most devastating tropical diseases, affecting about 250 million people around the world. For this reason, it is considered the second most important parasitic diseases in terms of socio-economic effects, losing only to malaria. In Brazil, Schistosoma mansoni is the only etiological agent of schistosomiasis, affecting 19 States. In areas of recent disease introduction or where the control occurs, low parasite burden is a reality and its detection is hampered by the low sensitivity of the available diagnostic methods. Helmintex? is a recently proposed method that uses a large amount of feces, numerous concentration steps, and addition of paramagnetic particles, conveying high sensitivity for the detection of eggs of S. mansoni in feces. However, it is a laborious process, with limited application in the field and delayed delivery of the diagnosis. The objective of this thesis is the improvement of egg detection methodologies for the diagnosis of schistosomiasis, mainly seeking the improvement of the Helmintex? methodology. Thus, the first chapter of this thesis proposes the reduction of the final Helmintex? sediment, with the use of neutral detergent, Tween 20, and the staining of S. mansoni eggs with ninhydrin, aiming to optimize the methodology. The second chapter investigates the use of a chemiluminescent reaction for the detection of S. mansoni eggs in the final Helmintex? sediment, aiming to speed up the final diagnosis. Finally, the third chapter explores the peculiarities of eggs and their passage through the intestinal wall, observing their clustering and the secretion by the eggs of a protein called Sm 16. The results obtained in this work reinforce the proposed role of the Helmintex method, to serve as a reference for the diagnosis of intestinal schistosomiasis, and for the comparative evaluation of other tests, in addition to registering for the first time, the formation of cluster and the excretion of the protein Sm16 by S. mansoni eggs during their migration through the intestinal wall of mice. Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES

A esquistossomose ? end?mica em 74 pa?ses, afetando mais de 200 milh?es de pessoas. No Brasil, o Schistosoma mansoni ? o ?nico agente etiol?gico da esquistossomose, que atinge 19 estados. Medidas de controle e tratamento efetivo podem resultar em diminui??o da carga parasit?ria de indiv?duos com a forma severa da doen?a. Assim, quando n?o forem utilizados m?todos diagn?sticos com sensibilidade adequada para acompanhamento, essas medidas podem dificultar o diagn?stico, favorecendo a perman?ncia da infec??o por longos per?odos, al?m da contamina??o ambiental e consequentemente a exposi??o ? popula??o local ? reinfec??o. Nestas ?reas, ou onde ocorreu introdu??o recente de S. mansoni, os m?todos parasitol?gicos cl?ssicos para encontrar ovos nas fezes n?o demonstram efic?cia. Embora as t?cnicas imunol?gicas e moleculares sejam consideradas atualmente as principais ferramentas de diagn?stico, s?o necess?rios m?todos parasitol?gicos para a confirma??o da infec??o. Recentemente, o Helmintex? foi descrito como um m?todo diagn?stico altamente sens?vel que isola ovos a partir de 30 gramas de fezes atrav?s da intera??o dos ovos com microesferas paramagn?ticos em um campo magn?tico. Apesar da sensibilidade, sua limita??o est? no n?mero de l?minas a serem analisadas, tornando o processo demorado e invi?vel para aplica??o na rotina cl?nica. Neste contexto, este trabalho abre perspectivas para a utiliza??o de novas ferramentas na tentativa de otimizar a detec??o de ovos de S. mansoni na ?ltima etapa do m?todo Helmintex?, utilizando a quimioluminesc?ncia, e a colora??o por ninidrina. Outra alternativa testada foi a colora??o do sedimento final com ninidrina. Os sedimentos de Helmintex? contendo ovos de S. mansoni foram fixados com etanol 70 % e depositados em papel filtro Whatman Grau 541, imersos em uma solu??o de ninidrina:etanol (30:70%) para posterior visualiza??o dos ovos corados em microsc?pio ?ptico. O tempo de leitura de cada filtro foi cronometrado. As condi??es ideais para incuba??o com a ninidrina foram estabelecidas em 15 minutos ? 24 ?C. O tempo de leitura para cada amostra foi, em m?dia, de 23 minutos. Em compara??o com o m?todo Helmintex? original, a utiliza??o de ninidrina diminuiu o tempo de leitura do sedimento final em, ao menos, 450 minutos. Os resultados deste trabalho mostraram que a implementa??o de novas ferramentas em m?todos diagn?sticos j? existentes podem contribuir para um melhor desempenho de t?cnicas sens?veis, por?m com aplica??o limitada em estudos de campo, e abrem perspectivas para novos estudos que visam investigar e otimizar etapas primordiais de m?todos de concentra??o. Schistosomiasis is endemic in 74 countries, affecting more than 200 million people. In Brazil, Schistosoma mansoni is the only causative agent of schistosomiasis, which affects 19 states. Control measurements and effective treatment can result in a decrease of the parasitic load of individuals with the severe form. In this matter, when diagnostic methods with adequate sensitivity for monitoring are not used, those measurements may difficult diagnosis, favoring the persistence of the infection for long periods, as well as the environmental contamination and consequently exposure to local population reinfection. In these areas, or where there was recent introduction of S. mansoni, classic parasitological methods to find eggs in feces are not efficient. Although immunological and molecular techniques are currently considered as main diagnostic tools, parasitological methods re necessary to confirm the infection. Helmintex? has been recently described as highly sensitive diagnostic method that isolates eggs from 30 gram of feces through the interaction of eggs with paramagnetic microspheres in a magnetic field. Despite the sensibility, its limitation lies in the number of slides to be analyzed, making the process time consuming and not applicable for clinical routine. In this context, this work opens perspectives for the use of new tools in an attempt to optimize S. mansoni egg detection in the last step of the Helmintex?, using chemiluminescence, and ninhydrin staining. Another alternative tested was the ninhydrin staining. Helmintex? sediments containing S. mansoni eggs were fixed with 70% ethanol and deposited on Whatman Grade 541 filter paper, immersed in a solution of ninhydrin:ethanol (30: 70 %) for later eggs visualization at the optical microscope. Reading time for each filter was counted and registered. Ideal conditions for incubation with ninhydrin were established in 15 minutes at 24 ?C. Reading time for each sample was, in general, of 23 minutes. When compared to the original Helmintex? method, the use of ninhydrin decreased the reading time of the final sediment in, at least, 450 minutes. The results of this work showed that the implementation of new tools in existent diagnostic methods may contribute to a better performance of sensitive techniques with limited application in field studies, and open perspectives to new studies that aim to investigate and to optimize primordial steps of concentration methods.

Paper presented at the 10th International Conference on the Scientific and Clinical Applications of Magnetic Carriers that took place in Dresden (Germany) during June 10-14th, 2014., Schistosomiasis is a public health problem affecting more than 200 million people in Asia, Africa and America. Two main species may cause the intestinal infection in humans: Schistosoma mansoni and Schistosoma japonicum. Helmintex® is a new very sensitive method for detection of Schistosoma eggs in human faeces based on the interaction of eggs and paramagnetic microspheres, with 100 % sensitivity at limit of 1.3 eggs per gram. The objective of this study was to investigate the magnetic properties of Schistosoma eggs and the interaction of microspheres with the eggs to enable optimization of the Helmintex® method. Eggs from both species were isolated from livers of infected mice and separately incubated with four types of microspheres at pH7 and pH 8 at an egg/microsphere ratio of 1:500 for 30 minutes with no applied magnetic field. The polystyrene microspheres were a) magnetic iron oxide coated; b) magnetic iron oxide and streptavidin coated; c) uncoated, d) streptavidin coated. The conjugates were sieved to remove unbound microspheres. An optical microscope was used to determine the distribution of the numbers of microspheres bound per egg. The observed distributions were well modeled with double Poisson distributions. At pH 7, both theS. japonicum and S. mansoni eggs appeared to fall into two types, one type having a greater affinity for magnetic iron oxide coated spheres than the other. S. japonicum eggs had a higher affinity for magnetic iron oxide coated microspheres than S. mansoni. Strepdavidin coating reduced the affinity for both species. At pH 8, the affinities of both species of eggs for the magnetic microspheres was reduced. In the absence of magnetic iron oxide coating, there was very little affinity of the eggs for microspheres. These observations suggest that the interaction between the microspheres and eggs is more likely to be related to electrostatic interactions between eggs and magnetic iron oxide rather than through magnetic interactions.