Dans la cellule hôte, Toxoplasma gondii se développe dans une vacuole parasitophore (VP) caractérisée par un réseau de nanotubes membranaires (RNM) dont la composition, le mécanisme de formation et la fonction sont obscures. Quelques protéines GRA, dont GRA2 et GRA6, sont sécrétées dans la VP à partir des granules denses puis ciblées au RNM. Cette localisation s'accorde avec l'hélice alpha-hydrophobe de GRA6 et les hélices alpha-amphipathiques de GRA2. Avant et après sécrétion dans la VP, les protéines GRA sont partiellement solubles. Le phénotype de parasites délétés de leur(s) gène(s) GRA2 et/ou GRA6 révèle que ces 2 protéines sont indispensables à la formation du RNM. J'ai montré 1) qu'avant leur insertion dans les membranes de la VP, la solubilité des protéines GRA est préservée grâce à des interactions hydrophobes avec peut être, des micelles de l'espace vacuolaire ; 2) que GRA12, une nouvelle protéine du RNM, n'interagit pas avec GRA2 dans ces membranes. 3) que l'adressage spécifique de GRA6 au RNM est déterminé par son domaine N-terminal hydrophile. 4) J'ai montré que GRA2 recombinante a une affinité pour le phosphatidyl inositol (4, 5) diphosphate avec lequel elle interagit via ses hélices alpha-amphipathiques. GRA2 déforme des liposomes de courbure membranaire importante pour générer de courts tubules membranaires. La tubulation est accentuée par GRA6 qui s'associe aux liposomes, quelque soit leur diamètre. Ces résultats valident le rôle direct de GRA2 et GRA6 dans la formation du RNM et laissent envisager un modèle de sa formation, dans lequel GRA6 favoriserait l'assemblage de vésicules lipidiques que GRA2 fusionnerait en tubules membranaires.

No description available.

Proteomics refers to global characterization of the full set of proteins present in a biological sample. Various analytical disciplines contribute to proteomics but mass spectrometry became method of choice for analysis of complex protein samples. Mass spectrometry allows for high throughput analysis of the proteome but, moreover, it has the ability to acquire higher-order information such as post-translational modifications (PTM). Glycosylation is the most abundant PTM on eukaryotic proteins. This dissertation will focus on method development for structural proteomics that will be utilized to explain the glycoproteome of obligate intracellular protozoan parasite Toxoplasma gondii as a model system. Optimization of sample preparation is addressed in the first part of this dissertation. Sample preparation for mass spectrometry analysis is a critical step in the proteomics workflow because the quality and reproducibility of sample extraction and preparation significantly impacts the separation and identification capabilities of mass spectrometers. Also, there are problems unique to intracellular parasites as limited amount, host cell impurity and choice of the host. The additional obstacle is to extract only glycosylated proteins for which there is no one standard method. Here we report the optimal sample preparation method utilizing agarose bound Concanavalin A (Con A) beads to efficiently pull down glycoproteins, dialyze and analyze them using MuDPIT. This method was further enhanced by passing the non-retained protein fraction (first flow-through) through a second Con A column and then passing the second non-retained protein fraction (second flow-through) through the third Con A column (3 sequential pull-downs) yielding 394 benchmark proteins. Glycoproteome of Toxoplasma gondii is not yet fully understood. However, evidence suggests that glycosylation could be essential for cyst formation and maintenance which is characteristic of chronic stage of disease. The focus of the second part of dissertation is to better understand the differences in glycoproteomes of tachizoites and tissue cysts. Cyst proteins pulled down using optimized sample preparation method that do not appear in the tachyzoites pulldowns could be critical elements in the structural stability of the tissue cyst.

The increasing prevalence of apicomplexan parasites such as Plasmodium, Toxoplasma, and Cryptosporidium represents a significant global healthcare burden. Treatment options are increasingly limited due to the emergence of new resistant strains. We postulate that parasites have evolved distinct metabolic strategies critical for growth and survival during human infections, and therefore susceptible to drug targeting using a systematic approach. I developed iCS306, a fully characterized metabolic network reconstruction of the model organism Toxoplasma gondii via extensive curation of available genomic and biochemical data. Using available microarray data, metabolic constraints for six different clinical strains of Toxoplasma were modeled. I conducted various in silico experiments using flux balance analysis in order to identify essential metabolic processes, and to illustrate the differences in metabolic behaviour across Toxoplasma strains. The results elucidate probable explanations for the underlying mechanisms which account for the similarities and differences among strains of Toxoplasma, and among species of Apicomplexa.

A survey of gastro-intestinal parasites was conducted on faecal samples collected from 379 feral cats and 851 native fauna from 16 locations throughout Western Australia. The prevalence of each parasite species detected varied depending upon the sampling location. Common helminth parasites detected in feral cats included Ancylostoma spp. (29.8%), Oncicola pomatostomi (25.6%), Spirometra erinaceieuropaei (14%), Taenia taeniaeformis (4.7%), Physaloptera praeputialis (3.7%) and Toxocara cati (2.6%). The most common protozoan parasites detected in feral cats were Isospora rivolta (16.9%) and I. felis (4.5%). The native mammals were predominately infected with unidentified nematodes of the order Strongylida (59.1%), with members of the orders Rhabditida, Spirurida and Oxyurida also common. Oxyuroid nematodes were most common in the rodents (47.9%) and western grey kangaroos (27.8%). Several species of Eimeria were detected in the marsupials whilst unidentified species of Entamoeba and coccidia were common in most of the native fauna. Primers anchored in the first and second internal transcribed spacers (ITS1 and ITS2) of the ribosomal DNA (rDNA) were used to develop a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) technique to differentiate the species of Ancylostoma detected in feral cats. Amplification of the ITS+ region (ITS1, ITS2 and 5.8S gene) followed by digestion with the endonuclease RsaI produced characteristic patterns for A. tubaeforme, A. ceylanicum and A. caninum, which were detected in 26.6%, 4.7% and 0% of feral cats respectively. Giardia was detected in a cat, dingo, quenda and two native rodents. Sequence analysis at the small subunit rDNA gene (SSU-rDNA) identified the cat and dingo as harbouring G.duodenalis infections belonging to the genetic assemblages A and D respectively. Subsequent analysis of the SSU-rDNA and elongation factor 1 alpha (ef1á) identified a novel species of Giardia occurring in the quenda. Attempts to genetically characterise the Giardia in the two native rodents were unsuccessful. Serological detection of Toxoplasma gondii was compared to a one tube hemi-nested PCR protocol to evaluate its sensitivity. PCR was comparable to serology in detecting T. gondii infections, although PCR was a much more definitive and robust technique than serology for large numbers of samples. Amplification of T. gondii DNA detected infections in 4.9% of feral cats and 6.5% of native mammals. The distribution of T. gondii does not appear to be restricted by environmental factors, which implies that vertical transmission is important for the persistence of T. gondii infections in Western Australia. These results demonstrate that cats carry a wide range of parasitic organisms, many of which may influence the survival and reproduction of native mammals. As such, the large-scale conservation and reintroduction of native fauna in Western Australia must not disregard the potential influence parasites can have on these populations.

Made available in DSpace on 2016-05-17T16:51:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-07-29. Added 1 bitstream(s) on 2016-05-17T16:54:50Z : No. of bitstreams: 1 000858047.pdf: 1402373 bytes, checksum: bba03f153ee64e0613ef1d472deb756d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A toxoplasmose e a leptospirose são doenças zoonóticas que acometem animais domésticos e selvagens. Inquéritos soroepidemiológicos em quatis (Nasua nasua) são importantes para conhecimento, vigilância e controle de doenças. O objetivo deste trabalho foi verificar a soroprevalência de anticorpos anti- Leptospira spp. e anti Toxoplasma gondii, em 108 amostras de soro de quatis mantidos em vida livre, provenientes do Parque Ecológico do Tietê, São Paulo, Brasil. Para a pesquisa de anticorpos anti-Toxoplasma gondii foi utilizado o teste de aglutinação modificada (MAT) verificando soroprevalência de 18 animais (16,67%) positivos e 90 animais (83,33%) negativos (IC 95%= 0,09- 0,23). Dos 18 animais positivos a frequência dos títulos apresentados foram: 25 (n=8; 44,44%), 50 (n=3; 16,67%), 100 (n=3; 16,67%), 200 (n=1; 5,56%) e 400 (n=3; 16,67%). Para pesquisa de anticorpos anti- Leptospira spp. foi utilizado o teste de Soroaglutinação Microscópica (SAM), sendo 96 animais negativos (88,89%) e 12 animais positivos, correspondente a soroprevalência de 11,11% (IC 95%= 6,0- 19), e os sorovares reagentes encontrados foram Butembo (n=2; 16,67%); Whitcombi (n=1; 8,33%); Sentot (n=1; 8,33%); Gryppotyphosa (n=1; 8,33%); Panama (n=1; 8,33%); Hardjo bovis (n=1; 8,33%); Hardjo ctg (n=1; 8,33%); Hardjo prajitno (n=4; 33,33%) e a frequência dos títulos apresentados foi: 100 (n=5; 41,67%); 200 (n=5; 41,67%); 400 (n=2; 16,67%). Portanto o presente estudo demonstra a importância da avaliação sorológica anti- Toxoplasma gondii e anti- Leptospira spp. em quatis de vida livre, o que avalia os aspectos epidemiológicos relacionados a estas zoonoses e riscos para a saúde pública, pois mantém contato com humanos / The toxoplasmosis and leptospirosis are zoonotic diseases that affect domestic and wild animals. Seroepidemiological surveys in coatis (Nasua nasua) is important for information, surveillance and disease control. The objective of this study was to determine the seroprevalence of antibody against Leptospira spp. and Toxoplasma gondii in 108 samples of coatis serum maintained in the wild, from the Ecological Park of Tietê, São Paulo, Brazil. For research of anti-Toxoplasma gondii antibodies we used the modified agglutination test (MAT) and 18 animals (16.67%) were soropositive and 90 animals (83.33%) negative (95% CI = 0,09- 0.23). Of the 18 positive animals positive the frequency of the antibodies were: 25 (n = 8; 44.44%), 50 (n = 3; 16.67%), 100 (n = 3; 16.67%), 200 (n = 1; 5.56%) and 400 (n = 3; 16.67%). To search the anti-Leptospira spp antibodies. It was used the Microscopic Agglutination Test test (MAT), and 96 were negative 88.89%) and 12 positive (11,11%) (95% CI = 6,0- 19). The serovars reagents Butembo (n = 2; 16.67%); Whitcombi (n = 1; 8.33%); Sentot (n = 1; 8.33%); Gryppotyphosa (n = 1; 8.33%); Panama (n = 1; 8.33%); Hardjo bovis (n = 1; 8.33%); Hardjo ctg (n = 1; 8.33%); Hardjo prajitno (n = 4; 33.33%). The frequency of the presented titles were 100 (n = 5; 41.67%); 200 (n = 5; 41.67%); 400 (n = 2; 16.67%). Therefore, this study demonstrates the importance of evaluating the serological response to anti- Toxoplasma gondii and for anti-Leptospira spp. of captive animals to assess epidemiological aspects related to these zoonoses and risks to public health because it maintains contact with humans

Made available in DSpace on 2014-06-11T19:32:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-14Bitstream added on 2014-06-13T20:04:31Z : No. of bitstreams: 1 lopes_wdz_dr_jabo.pdf: 1071597 bytes, checksum: d61e6ffa60481492021ac9df6eda129f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Ovinos machos isentos de Toxoplasma gondii, foram distribuídos em três grupos sendo, G1: um ovino inoculado, pela via oral, com 2,0 x 105 oocistos da cepa P, G2: um ovino infectado, pela via subcutânea, com 1,0 x 106 taquizoítos da cepa RH e G3: um ovino não infectado mantido como controle. Após a inoculação dos machos com T. gondii, 12 ovelhas reprodutoras, não gestantes, sorologicamente negativas para doenças reprodutivas, sobretudo toxoplasmose, foram sincronizadas e em seguida expostas à monta natural pelos machos, anteriormente inoculados, sendo: cinco ovelhas submetidas à monta natural pelo macho do G1; cinco ovelhas expostas à monta natural pelo macho do G2 e duas ovelhas pelo macho pertencente ao grupo controle. Nos soros das ovelhas obtidos nos dias -30, -14, -7, -1, zero (antes da monta natural) e nos dias 1, 3, 5, 7, 11, 14 e semanalmente até o parto, foi pesquisada a presença de anticorpos contra T. gondii pela RIFI. Bioensaio em camundongos e PCR foram realizados em amostras de sêmen e tecidos dos machos, tecidos das fêmeas e de seus respectivos filhotes. Cinco das 12 fêmeas utilizadas apresentaram anticorpos específicos contra T. gondii após a monta natural, sendo duas pelo macho inoculado com oocistos (G1) e três pelo ovino infectado com taquizoítos (G2). Pelo bioensaio foi possível diagnosticar, no dia da monta natural, a presença do T. gondii em amostras seminais dos ovinos infectados (G1 e G2) e em amostras do “pool” de tecidos das cinco fêmeas e em cinco de seus respectivos filhotes. Foi possível, ainda, pela técnica da PCR isolar o DNA de T. gondii em amostras seminais dos machos reprodutores no dia do coito, e no “pool” de tecidos de uma e duas fêmeas expostas à monta natural por reprodutores infectados com oocistos e taquizoítos, respectivamente. É importante relatar que, por meio desta técnica foi possível diagnosticar... / Male sheep in reproductive age and with no prior Toxoplasma gondii disease, were divided into three groups: G1, a sheep inoculated with 2.0 x 105 of P strain oocysts; G2, a sheep infected with 1.0 x 106 of RH strain tachyzoites and; G3 a control uninfected sheep. After inoculation of males with T. gondii, 12 breeding ewes, not pregnant, serologically negative for reproductive diseases, particularly toxoplasmosis, were synchronized and then exposed to natural mating by those males, previously inoculated, being: five ewes submitted to natural mating by male G1, five ewes exposed to natural mating by the male of G2 and two ewes submitted to natural mating by the male control from group. In sera obtained from all ewes on days -30, -14, -7, -1, zero (before natural mating) and on days 1, 3, 5, 7, 11, 14 and weekly until partum, was investigated the presence of antibodies against T. gondii by IFAT. Bioassay and PCR were performed on samples of semen and tissues of males/females tissues and their offspring. Five of the 12 females submitted to natural mating, had antibodies anti-T. gondii, being, two by male inoculated with oocysts (G1) and three for sheep infected with tachyzoites (G2). For the bioassay was possible to diagnose on the day of natural mating, the presence of T. gondii in semen samples from infected sheep (G1 and G2) and in samples of pool of tissues from five females and five of their puppies. It was also possible by PCR to isolate the DNA of T. gondii in semen samples of rams on sexual intercourse, and the pool of tissues of one and two females exposed to breeding by natural mating infected with tachyzoites and oocysts, respectively. It is important to report that through this technique it was possible to diagnose the presence of this parasite also in the pool of tissue from the offspring of a female submitted to the natural breeding sheep infected with oocysts. From the results... (Complete abstract click electronic access below)

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-09-06T20:11:44Z No. of bitstreams: 1 MariaEduardaDeSouzaMenezesDaCosta_DISSERT.pdf: 2415680 bytes, checksum: c90f35fd25510d290113b437f4a11687 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-09-06T23:03:43Z (GMT) No. of bitstreams: 1 MariaEduardaDeSouzaMenezesDaCosta_DISSERT.pdf: 2415680 bytes, checksum: c90f35fd25510d290113b437f4a11687 (MD5) / Made available in DSpace on 2016-09-06T23:03:43Z (GMT). No. of bitstreams: 1 MariaEduardaDeSouzaMenezesDaCosta_DISSERT.pdf: 2415680 bytes, checksum: c90f35fd25510d290113b437f4a11687 (MD5) Previous issue date: 2016-01-27 / O Toxoplasma gondii ? um parasito mundialmente distribu?do, que pode causar desde sintomas parecidos com a gripe at? problemas neurol?gicos. As cepas do T. gondii apresentam grande variabilidade gen?tica na Am?rica do Sul, sendo fundamental a an?lise de sequ?ncias para auxiliar no diagn?stico confi?vel e para classifica??o dos diferentes gen?tipos. O objetivo deste trabalho foi desenvolver dois m?todos moleculares, um para detec??o e o outro para genotipagem do T. gondii, possibilitando a identifica??o de todas as cepas conhecidas e a gera??o de dados que possam ser correlacionados com a virul?ncia. Inicialmente, foi utilizado o programa MUSCLE para alinhamento de 685 sequencias nucleot?dicas obtidas do GenBank, em seguida os alinhamentos foram analisados no programa MEGA 6.0 para determina??o de sua variabilidade gen?tica. O gene GRA7 foi selecionado como alvo para os iniciadores, que foram desenhados em regi?es conservadas do gene utilizando os programas Geneious 9.0 e Primer BLAST. O protocolo de amplifica??o utilizando-se os primers para o gene GRA7 foi ent?o comparado com outros protocolos padronizados para amplifica??o do gene B1 e do elemento repetitivo de 529 pb, que s?o os marcadores mais utilizados para o diagn?stico do T. gondii. Para o desenvolvimento do sistema de genotipagem foram selecionados os genes ROP5, ROP18 e GRA7, que est?o relacionados ao mecanismo de virul?ncia melhor descrito em T. gondii. O sistema de genotipagem desenvolvido se baseia na an?lise de polimorfismos presentes em fragmentos sob sele??o positiva desses genes, que permite identificar cepas pertencentes as linhagens clonais e cepas at?picas. Utilizando-se essa nova abordagem para a sele??o de marcadores, ser? necess?rio investigar um n?mero de regi?es do genoma consideravelmente menor que o utilizado pelo m?todo utilizado tradicionalmente, simplificando o processo. Concluindo, o desenho de primers em regi?es conservadas do gene GRA7 permitiu o desenvolvimento de um sistema de detec??o utilizando PCR com excelente positividade e sensibilidade, principalmente para detec??o de cepas at?picas do T. gondii. Ainda, a genotipagem baseada na detec??o de polimorfismos em genes de virul?ncia permitiu a diferencia??o genot?pica das diferentes cepas de forma mais simples que a t?cnica de RFLP utilizada atualmente.

Une immunité concomitante à long terme est mise en place lors d’infections persistantes avec des parasites protozoaires intracellulaires responsables, par exemple, de la leishmaniose et du paludisme. Dans un modèle murin de leishmaniose, il a ainsi été démontré que les cellules T régulatrices CD4+CD25+ sont impliquées dans la persistance des leishmanies aux sites d’infection cutanés et protègent l’hôte contre une ré-infection.Le protozoaire Toxoplasma gondii est également à l’origine d’une infection chronique liée à l’installation du parasite dans le cerveau et les muscles de l’hôte dans des formes kystiques. Il était donc pertinent de s’intéresser à l’implication des cellules T régulatrices dans l’installation et la persistance du toxoplasme.Pour atteindre cet objectif, nous avons utilisé l’anticorps monoclonal anti-CD25 dans des expériences de déplétion pendant la phase aiguë de la toxoplasmose après infection de souris non consanguines avec une souche de toxoplasmes de type II. Aucune différence significative que ce soit en terme de mortalité ou de charge parasitaire cérébrale n’a été observée entre les souris infectées et déplétées et les souris infectées non déplétées. En complément de ces expériences, nous avons pu montrer que les cellules régulatrices CD4+CD25+Foxp3+ (Treg) sont une cible potentielle de l’anticorps anti-CD25 ainsi que les cellules T effectrices CD4+CD25+Foxp3- (Teff); cellules qui expriment le marqueur CD25 en phase aiguë de l’infection. / Long term concomitant immunity is developed in case of persistant infections with intracellular protozoan parasites like for example in leishmaniosis and malaria. In a murine model of leishmaniosis, it has been demonstrated that CD4+CD25+ regulatory T (Treg) cells are involved in the persistance of leishmania parasites at cutaneous sites of infection and protect the host against re-infection.The protozoan parasite Toxoplasma gondii is also responsible for a chronic infection associated with the settlement of parasite in the brain and the muscles of the host in the form of cysts. It was therefore pertinent to know about the implication of Treg cells in the development and the persistance of toxoplasma. To attain this objective, we have used a monoclonal antibody anti-CD25 in depletion experiments during the acute phase of toxoplasmosis after infection of outbred mice with a type II toxoplasma strain. No significant difference was found in terms of mortality or in brain cyst load between depleted mice and non-depleted mice. In addition to these experiments, we have shown that not only the CD4+CD25+Foxp3+ regulatory T (Treg) cells but also the CD4+CD25+Foxp3- T effector (Teff) cells are a potential target of anti-CD25 antibody-depletion. These cells are induced to express CD25 during acute phase of the infection.

Introdução: A infecção aguda pelo Toxoplasma gondii em gestantes pode determinar infecção fetal através de passagem transplacentária. As crianças afetadas podem desenvolver coriorretinite e déficit neurológico, na ausência de tratamento adequado. Objetivos: Estimar a prevalência de toxoplasmose aguda em gestantes atendidas na maternidade do Hospital Nossa Senhora da Conceição, avaliando possíveis diferenças nas freqüências ao longo do período estudado; medir a incidência de toxoplasmose congênita (TC) e estimar a taxa de transmissão vertical em crianças nascidas neste hospital; avaliar a acurácia de testes diagnósticos em TC, aplicados no momento do nascimento, na população de crianças estudadas. Métodos: Inicialmente um estudo transversal foi desenvolvido para identificar as pacientes que apresentaram critérios de infecção aguda na gestação, atendidas na maternidade no momento do parto, entre outubro de 1998 e dezembro de 2005. Novas tecnologias foram introduzidas para detecção diagnóstica ao longo deste período. Entre outubro de 1998 e dezembro de 2001 (período 1), utilizou-se o método microparticle enzyme immunoassay – MEIA e entre janeiro de 2002 e dezembro de 2005 (período 2) foi utilizada a técnica de captura de IgM e o teste de avidez de IgG com o método enzyme linked fluorescent assay – ELFA (VIDAS). Os recém-nascidos identificados a partir deste estudo inicial foram incluídos em um estudo de coorte histórico, com tempo de acompanhamento aproximado de doze meses, para estabelecer o diagnóstico definitivo de toxoplasmose congênita, obtido em dois momentos: (1) logo após o nascimento; (2) aos 12 meses, aproximadamente. Para a definição de caso da infecção aguda na gestação e de toxoplasmose congênita foi utilizado o sistema de classificação elaborado por Lebech et al., com adaptações (quadro 1). Aqueles casos classificados como improváveis foram excluídos do cálculo da prevalência de toxoplasmose aguda em gestantes e de incidência de TC. Para avaliar a acurácia de testes diagnósticos em TC realizados no momento do nascimento utilizou-se, como padrão ouro para o diagnóstico definitivo, a concentração de anticorpos IgG em torno de 12 meses de vida. Um bebê infectado deve ter concentrações iguais ou mais elevadas de IgG nesta idade quando comparadas às encontradas no nascimento. Houve comparação independente e cega dos métodos diagnósticos avaliados em relação ao padrão ouro. A dosagem de anticorpos IgG e IgM em recém-nascidos foi processada com o método microparticle enzyme immunoassay (MEIA). Análise: Para comparar proporções foi utilizado o teste qui-quadrado com correção de Yates, teste exato de Fisher, quando necessário, e Teste t de Student para comparação entre médias de duas amostras independentes com distribuição simétrica. Na avaliação do desempenho de testes diagnósticos foram obtidos resultados de sensibilidade, especificidade, razões de verossimilhança (RV) positiva e negativa dos testes diagnósticos – hemograma, exame do líquor, ecografia transfontanelar, exame de fundo de olho (EFO) e a detecção de anticorpos IgM para toxoplasmose (MEIA) e do DNA do toxoplasma com reação em cadeia da polimerase – método Nested (RCP-Nested) séricos. Resultados: Em uma população de 41.112 gestantes, a prevalência de toxoplasmose aguda foi de 4,8 para cada 1.000 gestantes (IC95%: 4,2 a 5,6). Houve redução significativa na prevalência de toxoplasmose aguda nas gestantes deste hospital a partir do ano 2002 (P=0,008). O diagnóstico de toxoplasmose congênita foi definitivo em 37 crianças entre 40.727 nascidos vivos no período estudado, atingindo uma incidência de 0,9 para cada 1.000 nascimentos (IC95%: 0,6 a 1,3). Logo após o nascimento, entre os 200 recém-nascidos expostos ao T. gondii, resultado de 199 gestantes infectadas, 25 bebês apresentaram critérios diagnósticos de toxoplasmose congênita, atingindo taxa de transmissão vertical de 12,5% (IC 95%: 8,2% - 17,9%). Após o seguimento foram detectados mais 12 casos, aumentando esta taxa para 18,5% (IC 95%: 13,4% – 24,6%). Para avaliar a acurácia de testes diagnósticos aplicados no momento do nascimento, foram identificadas 31 crianças com TC, de acordo com o padrão ouro, entre 136 expostas ao protozoário intra-útero e que completaram seguimento até os 12 meses de vida. Os testes que apresentaram melhor desempenho isoladamente na predição do diagnóstico, foram detecção de anticorpos IgM específicos, EFO e RCPNested atingindo razões de verossimilhança positivas de 119,3 (IC95%: 7,40 – 1923,92), 49,9 (IC95%: 3,0 – 838,2) e 24,8 (IC95%: 3,22 – 190,35), respectivamente. A RV negativa para IgM foi 0,4 (IC95%: 0,3 – 0,6), mas para o EFO e RCP-Nested foi apenas 0,7 (IC95%: 0,6 – 0,9). Conclusão: A prevalência de toxoplasmose aguda em gestantes incluídas neste estudo foi inferior à encontrada na França e na Bélgica, mas foi mais elevada quando comparada às descritas na Suécia, Noruega, Dinamarca e Nova Iorque. A prevalência estimada neste estudo foi similar a de outros locais do Brasil, como Mato Grosso do Sul, mas inferior à obtida no Distrito Federal. Esta variabilidade nas estimativas pode estar relacionada com os diferentes métodos diagnósticos utilizados no rastreamento, ou ainda, com os diferentes fatores de risco envolvidos na transmissão da doença. Em nosso estudo, esta freqüência diminuiu a partir do ano de 2002, o que não pode ser atribuível à introdução do teste de avidez de IgG, uma vez que a média de idade gestacional na realização deste exame foi tardia. Entretanto, existe a possibilidade de que o teste de captura de IgM com o método ELFA tenha contribuído para diminuir os casos falso-positivos de IgM, o que poderá ser confirmado com futuros estudos. Estes resultados apontam que a introdução de novas tecnologias no laboratório deve ser acompanhada de esforços para melhorar as condições de acesso precoce das gestantes ao pré-natal de referência, com o objetivo de não perder a oportunidade para melhor discriminar as gestantes sem doença e evitar procedimentos invasivos, desnecessários e onerosos. A incidência de toxoplasmose congênita e a taxa de transmissão vertical foram elevadas. Estas freqüências podem estar subestimadas devido às perdas no seguimento. Para avaliar o efeito das perdas na validade do estudo, foram comparadas as características da população de gestantes e de recém-nascidos do grupo de bebês com seguimento completo em relação ao grupo que não retornou para o seguimento. Estas características foram semelhantes nos dois grupos, portanto, consideramos que o percentual de perdas, embora elevado, não prejudicou a validade do estudo. A identificação de 12 casos adicionais de TC com o seguimento dos bebês reforça a necessidade de monitoramento sorológico durante o primeiro ano de vida, mesmo sem evidência de infecção congênita ao nascimento. Na avaliação dos testes diagnósticos aplicados ao nascimento, a detecção de anticorpos IgM específicos no sangue do neonato, o EFO e o RCP-Nested sérico demonstraram melhor desempenho para identificar os bebês com TC. Entretanto, para afastar este diagnóstico, apenas o resultado não reagente de anticorpos IgM específicos apresentou maior utilidade, mas com efeito de pequena magnitude. / Introduction: The acute infection by Toxoplasma gondii in pregnant women may cause fetal infection by means of the transplacental transfer. Affected children may develop chorioretinitis and neurological deficit in the absence of a proper treatment. Objectives: To estimate the prevalence of acute toxoplasmosis in pregnant women cared for at the maternity ward of Hospital Nossa Senhora da Conceição, evaluating possible differences in frequencies along the period of study; to measure the incidence of congenital toxoplasmosis (CT), and estimate the rate of vertical transmission in children who were born in this hospital; to evaluate the accuracy of diagnostic tests in CT, applied at the moment of the birth, in the population of studied children. Methods: Initially a cross-sectional study was developed to identify the patients who presented criteria of acute infection in the pregnancy, cared for at the maternity ward at the moment of the labor, between October 1998 and December 2005. New technologies have been introduced for diagnostic detection along this period. Between October 1998 and December 2001 (period 1) the microparticle enzyme immunoassay – MEIA was used, while between January 2002 and December 2005 (period 2) the IgM capture technique and the IgG avidity test with the enzyme linked fluorescent assay – ELFA (VIDAS) method were used. Those newborns (NB) identified at this initial study were included in a historical cohort study, with an approximate follow-up time of twelve months, to establish the definitive diagnosis of congenital toxoplasmosis, obtained in two moments: (1) soon after birth; (2) at 12 months, approximately. For the definition of cases of acute infection in pregnancy and congenital toxoplasmosis, the classification system elaborated by Lebech et al. was used with adaptations (Picture 1). Those cases that were classified as unlikely were excluded from the prevalence calculation of acute toxoplasmosis in pregnant women and from the incidence of CT. In order to evaluate the accuracy of diagnostic tests in CT accomplished at the moment of birth, the concentration of IgG antibodies around 12 months of age was used as a gold standard for the ultimate diagnosis. An infected baby must have equal or higher concentrations of IgG at this age when compared to the ones found at birth. An independent and blind comparison of the diagnostic methods evaluated in relation to the gold standard was performed. The dosage of IgG and IgM antibodies in newborns was processed with the microparticle enzyme immunoassay (MEIA) method. Analysis: In order to compare proportions, a chi square test with Yates correction, an exact Fisher test when necessary, and a Student's t-test for comparison between means of two independent samples with symmetrical distribution were used. In the performance evaluation of diagnostic tests, results of sensitivity, specificity, positive and negative likelihood ratio (LR) of the diagnostic tests – hemogram, liquor test, transfontanellar ultrasonography brain scan, ophthalmoscopy, and detection of IgM antibodies for toxoplasmosis (MEIA) and the DNA of the toxoplasm with polymerase chain reaction – method Nested (PCR-Nested) serum were obtained. Results: In a population of 41,112 pregnant women, the prevalence of acute toxoplasmosis was 4.8 per 1,000 pregnant women (CI95%: 4.2 to 5.6). There was a significant decrease in the prevalence of acute toxoplasmosis in pregnant women of this hospital from 2002 on (P=0.008). The diagnosis of congenital toxoplasmosis was definitive in 37 children among 40,727 live births in the studied period, reaching an incidence of 0.9 per 1,000 births (CI95%: 0.6 to 1.3). Soon after the birth, among the 200 newborns exposed to the T. gondii, resulting from 199 pregnant women, 25 babies showed diagnostic criteria of congenital toxoplasmosis, reaching a vertical transmission rate of 12.5% (CI 95%: 8.2% - 17.9%). After the follow-up, another 12 cases were detected, increasing this rate to 18.5% (CI 95%: 13.4% – 24.6%). In order to evaluate the accuracy of diagnostic tests applied at the moment of birth, 31 children with CT were identified according to the gold standard, among 164 ones who were exposed to protozoan intra-uterus and who completed their follow-up until 12 months of age. The tests that showed better performance when isolate in the prediction of the diagnosis were the detection of specific IgM antibodies, ophthalmoscopy, and PCR-Nested reaching positive RV of 119.3 (CI95%: 7.40 – 1923.92), 49.9 (CI95%: 3.0 – 838.2), and 24.8 (CI95%: 3.22 – 190.35), respectively. The negative RV for IgM was 0.4 (CI95%: 0.3 – 0.6), but for ophthalmoscopy and PCR-Nested it was 0.7 (CI95%: 0.6 – 0.9) only. Conclusion: The prevalence of acute toxoplasmosis in pregnent women included in this study was lower than the one found in France and Belgium, but was higher when compared to the ones described in Sweden, Norway, Denmark, and New York. The estimated prevalence in this study was similar to the one from other locations in Brazil, such as Mato Grosso do Sul, but lower to the one obtained in Brasília. This variability in the estimates may be related to the different methods for diagnosis used in the screening or even to the different risk factors involved in the transmission of the disease. In our study, this frequency decreased from 2002 on, and this cannot be attributed to the introduction of the IgG avidity test, since the means of gestational age at the accomplishment of this test was late. However, there is a possibility that the IgM capture test with the ELFA method has contributed to decrease the frequency of false-positive cases of IgM, what can be confirmed with future studies. These results signal that the introduction of new technologies in the laboratory must be accompanied by efforts to improve conditions for an early access of pregnant women to the reference prenatal care, aiming to prevent the loss of opportunities of better discriminating pregnant women that are not sick and avoiding unnecessary and expensive invasive procedures. The incidence of congenital toxoplasmosis and the rate of vertical transmission were elevated. These frequencies may be underestimated due to the losses in the follow-up. In order to evaluate the effect of losses in the study validity, characteristics of the population of pregnant women and newborns belonging to the group of babies with full follow-up in relation to the group that did not return for the follow-up were compared. These characteristics were similar in the two groups, therefore we consider that the percentual of losses, even though high, did not endanger the validity of the study. The identification of twelve additional cases of congenital toxoplasmosis with the follow-up of the babies reinforces the need of a serological monitoring during the first year of life, even without any evidence of congenital infection at birth. In the evaluation of diagnostic tests applied at birth the detection of specific IgM antibodies in the blood of the newborn, the ophthalmoscopy, and the serum PCR-Nested showed a better performance to identify babies with CT. However, in order to discard this diagnosis, only the non-reagent IgM specific antibodies result showed higher utility, with a small magnitude effect though.