Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:48:40Z No. of bitstreams: 1 Jaqueline Silva Cruz Avaliação...2014.pdf: 1187799 bytes, checksum: fc9ff7db8754a8ac356e1e5a26370207 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-05-12T13:48:50Z (GMT) No. of bitstreams: 1 Jaqueline Silva Cruz Avaliação...2014.pdf: 1187799 bytes, checksum: fc9ff7db8754a8ac356e1e5a26370207 (MD5) / Made available in DSpace on 2015-05-12T13:48:50Z (GMT). No. of bitstreams: 1 Jaqueline Silva Cruz Avaliação...2014.pdf: 1187799 bytes, checksum: fc9ff7db8754a8ac356e1e5a26370207 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A dengue é atualmente um dos principais problemas de saúde pública do mundo e segundo a Organização Mundial de Saúde (OMS) é a doença que mais acomete o homem na atualidade. Sua incidência vem aumentando e estima-se que 50-100 milhões de pessoas desenvolvam a doença a cada ano no mundo. O diagnóstico laboratorial da dengue é realizado por diferentes tipos de testes, entre eles estão o isolamento viral, o RT-PCR, e a detecção por ELISA ou por meio de testes rápidos do antígeno viral NS1 e de anticorpos IgM específicos contra o vírus. A fim de contribuir para um melhor entendimento sobre a validade destes testes em diferentes circunstâncias, o objetivo principal deste trabalho foi avaliar a validade dos diferentes métodos laboratoriais no diagnóstico da dengue. A sensibilidade dos testes diagnósticos, ELISA IgM, ELISA NS1 e RT-PCR, foi avaliada de forma individual e de forma combinada utilizando amostras de soro de 623 pacientes incluídos em um estudo prospectivo de vigilância de base populacional entre fevereiro e julho de 2010. A sensibilidade destes testes também foi avaliada de acordo com a duração dos sintomas, com o tipo de infecção (primária vs secundária) e, por sorotipo infectante. A especificidade de cada método foi avaliada em um grupo de amostras de pacientes com diagnóstico laboratorial de leptospirose, hepatite, doadores de sangue e indivíduos sadios. Os resultados encontrados mostraram que 240 (38%) dos pacientes com doença febril aguda apresentaram dengue no período do estudo sendo que 194 (81%) dos pacientes com dengue representavam pacientes com infecções secundárias, o sorotipo predominante foi o DENV-2 (70%). As sensibilidades do RT-PCR, do ELISA NS1 e do ELISA IgM na amostra de fase aguda foram de 83,3%, 31,7% e 30%, respectivamente. O uso combinado do teste RT-PCR e do teste ELISA IgM em uma amostra de fase convalescente foi capaz de identificar 100% dos casos confirmados de dengue. As especificidades encontradas variaram de 97% a 100% para o ELISA NS1 e de 55% a 85% para o ELISA IgM. Os resultados indicam que na fase aguda da doença o RT-PCR é mais sensível a detecção de anticorpos IgM e do antígeno NS1 por ELISA, entretanto, o uso de métodos diagnósticos adicionais pode ser necessário em pacientes com uma suspeita da doença e resultado negativo do RT-PCR. / Dengue is currently one of the main problems of public health and the world according to the World Health Organization (WHO) is the disease that affects more men today. Its incidence is increasing and it is estimated 50-100 million people develop the disease each year worldwide. Laboratory diagnosis of dengue is done by testing different types, which include viral isolation, RT-PCR, and detection by ELISA or by rapid viral tests NS1 antigen and specific IgM antibodies against the virus. In order to contribute to a better understanding of the validity of these tests in different circumstances, the aim of this study was to evaluate the validity of different laboratory methods for diagnosis of dengue. The sensitivity of diagnostic tests, IgM ELISA, ELISA NS1 and RT-PCR was evaluated individually and in combination form using serum samples from 623 patients enrolled in a prospective population-based study of surveillance between February and July 2010. The sensitivity of these tests was also evaluated according to the duration of symptoms of infection with the type (primary versus secondary), and the infecting serotype. The specificity of each method was evaluated in a group of samples from patients with laboratory diagnosis of leptospirosis, hepatitis, blood donors and healthy individuals. The results showed that 240 (38%) of patients with acute febrile disease had dengue during the study period of which 194 (81%) of patients with dengue represented patients with secondary infections, the predominant serotype was DENV-2 (70% ). The sensitivity of the RT-PCR of NS1 and IgM ELISA ELISA in the acute phase of the sample were 83.3%, 31.7% and 30%, respectively. The combined use of RT-PCR and ELISA IgM in a sample convalescent phase was able to identify 100% of confirmed cases of dengue fever. The specificities found varied from 97% to 100% for ELISA NS1 and 55% to 85% for the IgM ELISA. The results indicate that the acute phase of the disease the RT-PCR is more sensitive detection of IgM antibodies and NS1 antigen by ELISA, however, the use of additional diagnostic methods may be necessary in patients with a suspicion of disease and negative outcome of RT-PCR.

Le plasmocyte (PC), stade terminal de la différenciation du lymphocyte B induite par l'antigène, est la cellule effectrice de l'immunité humorale responsable de la production des anticorps. La population plasmocytaire se divise en deux grands sous-types différant par leur durée de vie et par leur localisation anatomique. On distingue ainsi les PC à durée de vie courte ou PC effecteurs (dans les tissus lymphoïdes secondaires) et les PC à longue durée de vi (LLPC ou PC à mémoire) localisés principalement dans la moelle osseuse. Ces derniers contribuent à la mémoire humorale en continuant à sécréter des anticorps protecteurs après résolution de l'infection. Nos résultats expérimentaux montrent que les Ags thymo-dépendants (TD) et les Ag thymo-indépendants (TI) induisent des PC médullaires exprimant des caractéristiques phénotypiques et fonctionnelles différentes. Ainsi, l'expression d'une forme membranaire fonctionnelle du récepteur antigénique (BCR) persiste sur les LLPC TI alors qu'elle est perdue sur les LLPC TD. Cette fonctionnalité nouvelle portée par les PC médullaires TI n'est pas dépendante de la sous population lymphocytaire B recrutée ni de la nature de l'Ag, mais de l'isotype IgM. Nos résultats montrent également que cette population de PC médullaires a les caractéristiques des LLPC : ces PC sont quiescents, ils demeurent dans la moelle osseuse jusqu'à 180 jours et sont phénotypiquement semblables aux LLPC. Nos travaux ont permis de montrer que les LLPC à IgM sont capables de reconnaître l'Ag et que l'engagement de leur BCR par l'Ag conduit à la production d'IL10 / Plasma cells (PC) represent the terminal differentiation stage of B lymphocytes. Their canonical function is to secrete antibodies (Abs). PC differentiation is driven by remodeling of the B cell transcriptional program, highlighted by the induction of the transcriptional repressor Blimp-1 and repression of Pax5, considered as the guardian of B cell identity. The dogma holds that PC, as opposed to B cells, have lost the Ag recognition capacity because they have switched from expression of a membrane-bound Ag receptor (mBCR) to production of the secreted form of the BCR (Abs). Here, we have compared the phenotypical and functional attributes of memory PC generated by the T cell-dependent (TD) and T-cell independent (TI) forms of the hapten NP. Our data show that TI NP-specific bone marrow (BM) PC generated by NP-dextran retain an Ag-binding capacity comparable to that of B cells long after immunization while TD NP-specific BM PC do not. We found that this difference is not imputable to the structure of the immunogen but is a specific feature of IgM-expressing PC, which are prominent in response to TI Ag. Upon Ag recognition in vitro, the mBCR of IgM+ BM PC promotes: i) Ca++ mobilization, ii) phosphorylation of Syk and Blnk, iii) Ag internalization and phosphorylation of the late endosomal kinase Erk. Finally, we demonstrate that Ag recall in vivo induces significant changes in the gene expression profile of NP-specific IgM+ BM PC with evidence for activation of a cytokine production program characterized in particular by up-regulation of the CCL5 and IL10 transcripts. In conclusion, our data show that IgM-expressing BM PC can sense Ag and may be driven to express a regulatory function upon Ag recall

Diagnosticering av neuroborrelios baseras på en kombinerad bedömning av neurologiska symtom, serologiska tester för Borrelia-specifika antikroppar och analys av cerebrospinalvätska för att räkna antalet vita blodkroppar och mäta nivåer av kemokinet CXCL13. Nyligen publicerade studier har visat på att analys av IgM-antikroppar riktade mot Borrelia inte är ett lika starkt laboratoriestöd som analys av IgG-antikroppar. Analys av IgM-antikroppar har bidragit till högre antal falskt positiva svar på grund av ospecifik reaktivitet till andra patogener och persisterande antikroppar från tidigare infektioner. Syftet med denna studie var att utvärdera det diagnostiska värdet av att analysera IgM antikroppar i cerebrospinalvätska och serumprover för patienter med misstänkt neuroborrelios. Dessutom jämfördes prestandan mellan två kemiluminiscens immunanalyser, LIAISON (DiaSorin, Italy) och VirClia (Vircell, Spain). För utvärderingen analyserades 80 patientprover på laboratoriet för Klinisk Mikrobiologi på Länssjukhuset Ryhov i Jönköping, Sverige.  För att konfirmera positiva resultatet från de två kemiluminiscens immunanalyserna användes immunoblot, EUROLINE (EUROIMMUN, Germany). Sammanfattningsvis finns det inte tillräckligt med bevis att analys av IgM har ett tillräckligt diagnostiskt värde för att bekräfta en misstänkt neuroborrelios-diagnos. Jämförelsen mellan två kemiluminiscens immunanalyser visade att VirClia kan vara ett lämpligare alternativ än LIAISON när få prover analyseras. / The diagnosis of neuroborreliosis is based on a combined evaluation of neurological symptoms, serological tests for Borrelia-specific antibodies, cerebrospinal fluid analysis to measure white blood cell count and chemokine CXCL13 levels. Recently published data has shown that analysis of IgM antibodies against Borrelia is not as supportive in establishing a clinical diagnosis as IgG antibodies. IgM analysis has contributed to higher false-positive rates because of unspecific reactivities to other biological agents and persistent antibodies from prior infections.  The purpose of this study was to assess the diagnostic value of analysing IgM antibodies in cerebrospinal fluid and serum samples from patients with suspected neuroborreliosis. The performance of two chemiluminescent immunoassays, LIAISON (DiaSorin, Italy) and VirClia (Vircell, Spain), were compared as well. For this assessment, 80 patient samples were analysed at the Laboratory of Clinical Microbiology in Jönköping County, Sweden. Immunoblotting, EUROLINE (EUROIMMUN, Germany), was utilised to validate positive results from the chemiluminescent immunoassays. In conclusion, there is no convincing evidence to suggest that IgM analysis has sufficient diagnostic value to confirm a suspected diagnosis of neuroborreliosis. The comparison of two immunoassays showed that VirClia could be a valid alternative to LIAISON when analysing fewer samples at a time.

Apoptose spielt eine wichtige Rolle bei der Generierung eines funktionellen Lymphozytenrepertoirs. Während der anti-IgM induzierten B-Zell Apoptose werden potentiell autoreaktive Zellen eliminiert. Da die molekularen Mechanismen der B-Zell Apoptose weitgehend unbekannt sind, sollten in dieser Arbeit assoziierte Proteine und Gene identifiziert werden. Durch Subklonierung wurde eine Burkitt Lymphom B Zellinie BL60-2 generiert, die sensitiv auf den anti-IgM Stimulus ist. Für den Vergleich apoptotischer und nicht apoptotischer Zellen wurde eine Methode entwickelt in der apoptotische, Phosphatidylserin (PS) positive Zellen, über eine magnetische Separation mit einer Reinheit von bis zu 95 Prozent von nicht apoptotischen Zellen angereichert werden konnten. Mittels der hochauflösenden 2D-Gelelektrophorese wurden die aufgereinigten Fraktionen apoptotischer und nicht apoptotischer Zellen analysiert und die Proteinmuster verglichen. Anhand massenspektrometrischer Analysen und Edman Abbau konnten bisher folgende differentiell erscheinende Proteinspots identifiziert werden: Lamin B1, b-Aktin, neutrales Calponin, Nucleolin, D4-GDI, hnRNP A1, hnRNP C1/C2, hnRNP K, LSP1, HHR23B, das FUSE-binding Protein, dUTPase, P0, HP1 a. Die Proteine D4-GDI, hnRNPA1 und der Transkriptionsfaktor SP1 werden spezifisch während der anti-IgM induzierten Apoptose gespalten. Der zeitliche Verlauf wurde in einer eindimensionalen Western Blot Analyse bestimmt. Das diese Spaltungen ein Resultat der Aktivierung der Proteasen der Caspase 3 Familie sind, konnte durch Inhibitor Studien mit dem Tetrapeptid z-DEVD-fmk bewiesen werden. Die Spaltung des Transkriptionsfaktors SP1 wurde ebenfalls in in vitro untersucht. Rekombinante Caspase 3 und 7 generieren die gleichen Spaltprodukte, die zuvor in vivo nach anti-IgM induzierter Apoptose detektiert wurden, Caspase 6 hingegen generiert nur ein Fragment. Die spezifische Spaltung des Transkriptionsfaktors beinflußt die DNA Bindungsaktivität, was in einem elektro mobility shift assay gezeigt werden konnte. Die Intensität des SP1/DNA-Komplexes nimmt nach Apoptose-Induktion stark ab, hingegen nimmt die Intensität eines kleineren Komplexes stark zu, was auf die Bindung eines der Spaltprodukte schließen ließ, das möglicherweise transkriptionell inaktiv ist und damit einen wichtigen Apoptose Effektor darstellt. Die Proteasen der Caspase 3 Familie konnten erstmalig als zentrale Regulatoren während der anti-IgM induzierten Apoptose identifiziert werden. Durch die Hemmung mit dem Inhibitor z-DEVD-fmk konnte die Apoptose, sowie charakteristische morphologische Veränderungen, wie die Kernfragmentierung und PS auf die Zelloberfläche gehemmt werden und als Caspase 3 abhängige Veränderungen bestimmt werden. Über verschiedene Methoden der differentiellen Hybridisierung von neu hergestellten l-Zap cDNA-Phagenbanken von nicht stimulierten und anti-IgM stimulierten Burkitt Lymphom Zellen sowie der subtraktiven Klonierung sollten Gene identifiziert werden, die während der anti-IgM induzierten Apoptose differentiell exprimiert werden. Apoptose spezifische Sequenzen wurden angereichert, kloniert und sequenziert. / Apoptosis or programmed cell death is essential in the process controlling lymphocyte growth and selection. During anti-IgM induced B cell apoptosis autoreactive cells are eliminated. Since the molecular mechanisms underlying the process of B cell apoptosis are still not well understood the goal of this study was to identify proteins and genes in apoptosis. Subcloning lead to the selection of a human Burkitt lymphoma B cell line clone (BL60-2) which is highly sensitive towards the anti-IgM stimulus. In order to compare apoptotic and non apoptotic cells a novel method was established, which allows separation of apoptotic, Phosphatidylserine (PS) positive cells from non apoptotic, PS negative cells by magnetic cell sorting. This method generates fractions of apoptotic and non apoptotic cells with a purity of up to 95 %. Cell lysates from those fractions were analyzed using high-resolution two-dimensional gel electrophoresis and the protein patterns were compared. Using mass spectrometry and Edman sequencing the following differentially appearing proteins were identified: Lamin B1, b-Actin, neutral Calponin, Nucleolin, D4-GDI, hnRNP A1, hnRNP C1/C2, hnRNP K, LSP1, HHR23B, FUSE-binding protein, dUTPase, P0, HP1 a. The specific cleavage of the D4-GDI, hnRNPA1 and the transcription factor SP1 which occurs after anti-IgM induced apoptosis was detected by western blot analysis. It was determined by inhibition studies with the tetrapeptide inhibitor z-DEVD-fmk that the cleavage is a result of the Caspase 3 family. Cleavage of the transcription factor SP1 was also investigated by in vitro cleavage assays. The activity of recombinant caspase 3 and 7 generate the same cleavage products as observed in vivo after anti-IgM induction. The specific cleavage of the transcription factor influences it´s DNA binding activity as observed by the decrease of the full lenght protein-DNA complex. The increase of a smaller protein-DNA complex was apparent, which may represent the binding of at least one cleavage product. In this work proteases of the Caspase 3 family could be identified as central regulators during anti-IgM induced apoptosis. Anti-IgM induced apoptosis can be completly inhibited by inhibition of Caspase 3 proteases. Characteristic morphological changes, nuclear fragmentation and PS translocation to the outer membrane leaflet of the cells could be determined as a consequence of the Caspase 3 activation during the process of apoptosis. By differential hybridisation of newly generated l-Zap c-DNA librarys from unstimulated and anti-IgM stimulated B cells as well as with subtractive cDNA librays, it should be feasable to identify newly transcribed genes. Apoptosis specific sequences were enriched, cloned and sequenced.

The innate immune protein surfactant protein (SP-) D is a carbohydrate binding protein that was originally isolated from mucosal lung tissues. Recently, studies show that SP-D binds to antibodies, including immunoglobulin M (IgM), which interacts with late apoptotic cells. Here we focus on the interaction between SP-D and IgM as they pertain to late apoptotic cell clearance. We hypothesized that the three-way interaction between IgM, SP-D and late apoptotic cells is functionally applicable to clearing late apoptotic cells from the lungs, thereby reducing lung inflammation. We show that SP-D binds to IgM and that IgM binds to the late apoptotic subclass of dying cells. We demonstrate that IgM and SP-D can both bind to late apoptotic cells in mutually distinct regions while also displaying some regional overlap. We show evidence that during LPS-induced lung inflammation both IgM and SP-D levels are elevated and this corresponds to an augmentation of apoptotic cell clearance. We illustrate that the protein interaction of IgM and SP-D is functionally relevant to apoptotic cell clearance in the lungs by showing that late apoptotic cells coated in IgM and/or SP-D are cleared more efficiently than control cells, by alveolar macrophages in vivo. Our ex vivo studies further show that these cells internalize apoptotic cells by engulfing very small particles released from the dying cells. We then showed that IgM preferentially directs the engulfment of small particles (~1 μm) by macrophages, in an apparent size-specific antibody-dependent particle clearance function. Our data reveals a novel relationship amongst IgM, SP-D, apoptotic cells, and alveolar macrophages that contributes to our understanding of apoptotic cell clearance, which may be used in the future to generate strategies addressing apoptotic cell accumulation or clearance deficiency in disease.

The innate immune protein surfactant protein (SP-) D is a carbohydrate binding protein that was originally isolated from mucosal lung tissues. Recently, studies show that SP-D binds to antibodies, including immunoglobulin M (IgM), which interacts with late apoptotic cells. Here we focus on the interaction between SP-D and IgM as they pertain to late apoptotic cell clearance. We hypothesized that the three-way interaction between IgM, SP-D and late apoptotic cells is functionally applicable to clearing late apoptotic cells from the lungs, thereby reducing lung inflammation. We show that SP-D binds to IgM and that IgM binds to the late apoptotic subclass of dying cells. We demonstrate that IgM and SP-D can both bind to late apoptotic cells in mutually distinct regions while also displaying some regional overlap. We show evidence that during LPS-induced lung inflammation both IgM and SP-D levels are elevated and this corresponds to an augmentation of apoptotic cell clearance. We illustrate that the protein interaction of IgM and SP-D is functionally relevant to apoptotic cell clearance in the lungs by showing that late apoptotic cells coated in IgM and/or SP-D are cleared more efficiently than control cells, by alveolar macrophages in vivo. Our ex vivo studies further show that these cells internalize apoptotic cells by engulfing very small particles released from the dying cells. We then showed that IgM preferentially directs the engulfment of small particles (~1 μm) by macrophages, in an apparent size-specific antibody-dependent particle clearance function. Our data reveals a novel relationship amongst IgM, SP-D, apoptotic cells, and alveolar macrophages that contributes to our understanding of apoptotic cell clearance, which may be used in the future to generate strategies addressing apoptotic cell accumulation or clearance deficiency in disease.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-18T19:05:14Z No. of bitstreams: 1 Andréia Carvalho dos Santos. Diagnostico sorologico...2011.pdf: 1884519 bytes, checksum: 6aff70f55415c00ce177ca938c909c5e (MD5) / Made available in DSpace on 2013-10-18T19:05:14Z (GMT). No. of bitstreams: 1 Andréia Carvalho dos Santos. Diagnostico sorologico...2011.pdf: 1884519 bytes, checksum: 6aff70f55415c00ce177ca938c909c5e (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A confirmação da leptospirose utilizando o Teste de Aglutinação Microscópica (MAT) requer amostras da fase aguda e convalescente para identificar soroconversão ou aumento de quatro vezes nos títulos. A Organização Mundial de Saúde (OMS) recomenda que a coleta da amostra convalescente seja realizada ≥14 dias após a coleta da amostra aguda. No entanto, a dificuldade na coleta de amostras convalescentes impede a confirmação dos casos e é uma das principais causas para sub-notificação da leptospirose. Este estudo investigou a viabilidade da coleta de uma amostra de soro aguda tardia de casos internados com leptospirose e avaliou se a análise sorológica desta amostra pode melhorar a eficiência do protocolo de confirmação diagnóstica de leptospirose. De 2003 a 2009, uma vigilância hospitalar ativa em Salvador-Brasil, identificou prospectivamente pacientes hospitalizados com suspeita clínica da leptospirose. Três amostras de sangue foram coletadas para cada caso: uma amostra aguda precoce, uma amostra aguda tardia e uma amostra convalescente, coletadas respectivamente nas primeiras 24 horas após hospitalização, e 4 e ≥14 dias depois da coleta da primeira amostra. Os pacientes identificados tiveram o diagnóstico de leptospirose confirmado por soroconversão, aumento de quatro vezes de títulos, ou título único ≥1:800 no MAT. O desempenho diagnóstico do MAT e do ELISA IgM na avaliação combinada das amostras aguda precoce e aguda tardia foi comparado ao desempenho da avaliação das amostras aguda precoce e convalescente que segue a recomendação de testagem da OMS. Nós confirmamos 643 (68%) dos 938 casos suspeitos. A coleta de amostra convalescente foi possível para 63% dos pacientes confirmados, e 55% dos pacientes suspeitos. Em contraste, a amostra da fase aguda tardia foi coletada para 77% e 66% dos pacientes confirmados e suspeitos, respectivamente. Para os 302 casos confirmados que tiveram as três amostras de soro coletadas, a sensibilidade do MAT e do IgM-ELISA na análise das amostras aguda precoce e tardia foi de 97% (IC95%, 94-99%) e 96% (93-98%), respectivamente, em comparação aos resultados da análise das amostras aguda precoce e convalescente. Em contraste, considerando apenas as amostras agudas destes 302 pacientes, a sensibilidade do MAT e do IgM-ELISA foi de 44% (38-50%) e 75% (69-79%), respectivamente. Amostra aguda tardia e convalescente foi obtida dos casos suspeitos de leptospirose que evoluíram para óbito de 32% e 6%, respectivamente. Os resultados indicam que a coleta e o teste sorológico da amostra aguda tardia de pacientes hospitalizados por leptospirose é viável e melhora a eficiência dos atuais protocolos de confirmação laboratorial de casos de leptospirose. / Confirmation of leptospirosis with MAT requires evaluating acute and convalescent-phase sera samples to identify seroconversion or fourfold rise in titers. Current World Health Organization (WHO) protocols recommend that convalescent samples are collected with ≥14days after the acute sample collection. However, the difficulty in collecting convalescent samples hampers case confirmation and is a major cause for leptospirosis under-reporting. This study evaluated feasibility of collecting a late acute-sera sample from hospitalized cases of leptospirosis and determined to serological analysis of this sample can improve the efficiency of the protocol to confirm the diagnosis of leptospirosis. From 2003 to 2009, active hospital-based surveillance in Salvador-Brazil prospectively identified hospitalized cases of patients with clinical suspicion of leptospirosis. Three blood samples were collected for each case: an early acute sample, a sample of late acute and convalescent sample collected during the first 24 hours after hospitalization 4 and ≥ 14 days after the first sampling, respectively. The identified patients were diagnosed with leptospirosis by seroconversion, fourfold rise in titers, or a titer ≥1:800 in the MAT. The diagnostic performance of the MAT and IgM ELISA in the combined sample of early acute and late acute sample performance was compared to the early assessment of acute and convalescent samples following a WHO recommendation for testing. We confirmed the leptospirosis diagnosis in 643 (68%) of 938 suspected cases. Convalescent-phase samples were collected from 63% of the confirmed patients, but in only 55% of the suspected cases. In contrast, the late acute phase sample was collected for 77% and 66% of confirmed and suspected patients, respectively. Among the 302 confirmed cases which all three samples were obtained, the sensitivity of MAT and IgM-ELISA was 97% (IC95%, 94-99%) and 96% (93-98%), respectively, when results of early and late acute-phase samples were evaluated in comparison to the results of the early acute and convalescent samples. In contrast, the sensitivity of MAT and IgM-ELISA was 44% (38-50%) and 75% (69-79%), respectively, when only a single early acute-phase sample was evaluated. Late acute-phase and convalescent-phase samples were obtained from 32% and 6% of the suspected leptospirosis and deaths, respectively. These findings indicate that collection and serologic testing of a late-acute-phase sample among hospitalized patients with suspected leptospirosis may significantly increase the efficiency of protocols for laboratory case confirmation.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-23T11:18:56Z No. of bitstreams: 2 Dissertação - Pedro Henrique Dias Botelho - 2017.pdf: 2156209 bytes, checksum: 7548059cd0e4b1077f097a09aa4fd4eb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-23T11:19:34Z (GMT) No. of bitstreams: 2 Dissertação - Pedro Henrique Dias Botelho - 2017.pdf: 2156209 bytes, checksum: 7548059cd0e4b1077f097a09aa4fd4eb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-23T11:19:35Z (GMT). No. of bitstreams: 2 Dissertação - Pedro Henrique Dias Botelho - 2017.pdf: 2156209 bytes, checksum: 7548059cd0e4b1077f097a09aa4fd4eb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-04-20 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Introduction. Laboratory tests are essential for dengue diagnosis, in that sense algorithms are proposed, which proposes instructions for a more effective laboratory dengue diagnosis. Aim. To evaluate the performance of laboratory tests in the confirmation of suspected dengue cases, appling an algorithm, during a dengue epidemic in Goiânia, Central West Brazil 2012-2013. Methodology. This is a retrospective analytical observational study in a database of a prospective cohort with suspected dengue cases. The algorithm applied was based on three periods in the acute phase of disease, 0-3, 4-7 and >7 days after onset of symptoms (DOS) and in detection of immunoglobulins M and G (IgM and IgG), non-structural 1 protein antigen (NS1Ag) and viral RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Positivity was seen individually and in association of tests in the algorithm and per day of infection, and used to confirm cases. The tests performance was evaluated by the sensitivity, specificity and accuracy of each test when compared to the others association, also in the algorithm. The results were statistically analyzed using SPSS Statistics 17.0, R software and OPENEPI. Results. 592 patients with suspected dengue were included, 415 (70.1%) were laboratory confirmed. In the 0-3 DOS period, the best positivities were by RT-PCR (81.6%) and NS1Ag (63.3%). While, IgM obtained the best positivities in 4-7 and >7 DOS periods (85.5% and 93.3%, respectively). Individually, RT-PCR and IgM tests were the most efficient to add positivity to diagnosis at the beginning and at the end of the acute phase of infection, respectively. Sensitivity results were similar to those of positivity, whereas NS1Ag specificities were greater than 90% at all periods. Conclusion. The algorithm sowed which laboratorial test was the best for the course of disease. Until 3 DOS, molecular is most sensitive test; between 4-7 DOS, two techniques may be required to obtain an accurate diagnostic. NS1Ag test, presented less detection in secondary infection cases, however, they was more specific test and can be used in differential diagnosis of dengue. These results contributed to diagnostic decision in the epidemiological context with concomitant arbovirus circulation. / Introdução. Testes laboratoriais são fundamentais para o diagnóstico da dengue, nesse sentido são propostos algoritmos, que propõe instruções para um diagnóstico laboratorial de dengue mais eficaz. Objetivo. Avaliar o desempenho de testes laboratoriais na confirmação de casos suspeitos de dengue, no curso de duas epidemias (2012 e 2013) em Goiânia, Goiás, Centro-Oeste do Brasil. Metodologia. Trata-se de um estudo observacional analítico que analisou uma base de dados clínicos e laboratoriais de uma coorte prospectiva de pacientes com suspeita clínica de dengue. O algoritmo aplicado baseou-se em três períodos da doença, 0-3, 4-7 e >7 dias após o início dos sintomas (DOS) e no uso de testes de detecção das imunoglobulinas M e G (IgM e IgG), do antígeno da proteína não-estrutural 1 (NS1Ag) e do RNA viral por reação em cadeia da polimerase via transcriptase reversa (RT-PCR). Foram avaliadas a positividade dos testes individualmente e em associação de testes por dia de infecção; e a sensibilidade, especificidade e acurácia dos testes. A análise estatistica usou os programas SPSS Statistics 17.0, R software e OPENEPI. Resultados. Dos 592 pacientes selecionados, 415 (70,1%) foram confirmados laboratorialmente. No período de 0-3 DOS, a RT-PCR e NS1Ag obtiveram 81,6% e 63,3% de positividade respectivamente. IgM obteve as positividade nos períodos de 4-7 e >7 DOS (85,5% e 93,3%, respectivamente). Individualmente, os testes de RT-PCR e IgM positividade ao diagnóstico no início e no final da fase aguda da infecção, respectivamente. Os resultados de sensibilidade foram semelhantes aos de positividade, enquanto os de especificidade de NS1Ag foram superiores à 90% em todos os períodos. Conclusão. O algoritmo apontou qual teste laboratorial foi o melhor para o curso da doença. Até 3 DOS, o teste molecular é o mais sensível; Entre 4-7 DOS, duas técnicas podem ser necessárias para obter um diagnóstico preciso. O teste de NS1Ag apresenta menor detecção em casos de infecção secundária, no entanto, foi o teste mais específico, podendo ser utilizado no diagnóstico diferencial de dengue. Estes resultados contribuíram para a decisão diagnóstica no contexto epidemiológico com a circulação concomitante de arbovírus.

Submitted by biblioteca unifenas (biblioteca@unifenas.br) on 2017-09-13T13:25:28Z No. of bitstreams: 1 Mauro Barbieri Dissertação.pdf: 1026519 bytes, checksum: b56b00d6a2720e47649bc500b977c098 (MD5) / Made available in DSpace on 2017-09-13T13:25:28Z (GMT). No. of bitstreams: 1 Mauro Barbieri Dissertação.pdf: 1026519 bytes, checksum: b56b00d6a2720e47649bc500b977c098 (MD5) Previous issue date: 2015-03-27 / The objective of the experiment evaluating different levels of egg powder residue in the diets of piglets weaned at 28 days of age, assessing performance, hematological parameters, the bacterial count and the quantification of immunoglobulin (IgM). The study was conducted at the Swine Federal Institute of Education Science and Technology of South of Minas Gerais Campus Muzambinho. One design was used in a randomized block design with four treatments and six replications and three animals per experimental unit. The initial weight was used as a criterion for constitution of blocks and treatments consisted of four powdered egg levels (0.0%, 2.0%, 4.0% and 6.0%). The results were evaluated using the linear regression analyzes and polynomial, and the adjustments evaluated for accuracy (R2). With the increase of egg powder residue levels there was an improvement in animal performance. In general, with increasing levels of powdered egg residue there was an increase in final weight of the animals, weight gain and an improvement in feed conversion. Regarding the blood components, there was a reduction on packed cell volume and total protein of seguimentados with raising the level of egg powder residue. The egg powder residue improves the performance of piglets weaned at 28 days of age. As one increases the inclusion of egg residue powder in the diet, there is a higher incidence of beneficial bacteria in tratogastrinteninal piglets, keeping them imonologicamente healthier / Objetivou-se, com a condução do experimento, avaliar diferentes níveis de resíduo de ovo em pó na dieta de leitões desmamados aos 28 dias de idade, avaliando o desempenho, os parâmetros hematológicos, a contagem bacteriana e a quantificação de imunoglobulinas (IgM). O trabalho foi desenvolvido na Suinocultura do Instituto Federal de Educação Ciência e Tecnologia do Sul de Minas Gerais Campus Muzambinho. Estabeleceu-se um delineamento em blocos ao acaso, com quatro tratamentos e seis repetições, sendo três animais por unidade experimental. Usou-se o peso inicial como critério para constituição dos blocos e os tratamentos foram constituídos por quatro níveis de ovo em pó (0,0%; 2,0%; 4,0% e 6,0%). Avaliaram-se os resultados por meio das das análises de regressão linear e polinomial e os ajustes, pela precisão (R2). Com o aumento dos níveis de resíduo de ovo em pó, houve melhora não só no peso final como também no ganho de peso médio diário e na conversão alimentar dos animais. Constatou-se redução do volume globular, dos segmentados e da proteína total com a elevação do nível de resíduo de ovo em pó. Além disso, com o aumento dos níveis do ovo, observou-se maior número de Bifidobacterium e Lactobacillus no cólon proximal dos leitões e menor contagem de Escherichia coli. O resíduo de ovo em pó melhora o desempenho dos leitões desmamados aos 28 dias de idade. À medida que se aumenta a inclusão de resíduo de ovo em pó na dieta, há maior incidência de bactérias benéficas no trato gastrointestinal dos leitões, mantendo-os imunologicamente mais saudáveis.

Cette these en cotutelle entre le Laboratoire Peptlab de l’Université de Florence en Italie et le Laboratoire de Pharmacognosie de l’Université de Bourgogne Franche-Comté en France, porte sur l’extraction, la purification et l’élucidation structurale de saponines d’origine végétale en tant que mimétiques d’antigènes impliqués dans une réponse immunitaire. L’étude phytochimique de cinq espèces végétales appartenant à trois familles différentes, Fabaceae : Wisteria frustecens, Wisteria floribunda “macrobotrys”, Wisteria floribunda “rosea”, Caprifoliaceae : Weigela florida “rumba” et Polygalaceae : Polygala acicularis, a conduit à l’isolement de seize glycosides triterpéniques naturels parmi lesquelles six sont de structure nouvelle, une a été isolée sous sa forme native pour la première fois, et neuf déjà répertoriées dans la littérature. Les composés ont été isolés grâce à l’utilisation de différentes méthodes chromatographiques. Leurs structures ont été élucidées en utilisant principalement la RMN 2D et la spectrométrie de masse. Parmis ces seize molécules, six ont été sélectionnées pour être testées en tant que mimétiques d’antigènes impliqués dans une réponse immunitaire. De plus, un flavonoïde glycosylé extrait de Sophora japonica et un acide triterpénique commercial, l’acide ursolique, ont eux aussi été choisis comme mimétiques d’antigènes. Des tests immunochimiques (ELISA) ont été réalisés afin d’évaluer leur potentiel en tant que mimétiques d’antigènes dans le sang de patients atteints de sclérose en plaque ou du syndrome de Rett. Le taux IgM dans le sérum des patients atteints de sclérose en plaque ou du syndrome de Rett a été mesuré et comparé à celui de donneurs sains. Concernant la sclérose en plaque, les résultats sont peu significatifs concernant le potentiel des saponines en tant que mimétiques d’antigènes. Mais dans le cas du syndrome de Rett des résultats intéressants et surprenants ont été obtenus. En effet, l’hypothèse de départ était l’implication de la partie glycosylée dans la reconnaissance d’autoanticorps. Pour le syndrome de Rett, l’acide ursolique, qui est un aglycone, démontre une grande efficacité dans la reconnaissance d’IgM. Par contre, un triterpène glycosylé démontre lui aussi une efficacité semblable. Les résultats obtenus sont donc à analyser afin d’établir des relations structure/activité fiables. / This PhD in co-direction between the Peptlab Laboratory of the University of Firenze (Italy) and the Laboratory of Pharmacognosy of the University of Bourgogne Franche-Comté (France), deals with extraction, purification and structural elucidation of saponins from plants as mimetic antigens involved in an immune response. The phytochemical study of five species from three different families, Wisteria frustecens, Wisteria floribunda “macrobotrys” and Wisteria floribunda “rosea” from Fabaceae, Weigela florida “rumba” from Caprifoliaceae, and Polygala acicularis from Polygalaceae, allowed us to isolate sixteen natural glycosides: six with new structures, one analyzed for the first time in its native form, and nine which have been already described in the literature. These compounds were isolated using various chromatographic methods, and their structures were elucidated using mainly 2D NMR and mass spectrometry. From the isolated glycosides, six were selected and tested as mimetics of native antigens involved in the immune response. Moreover, one flavonoid glycoside extracted from Sophora japonica, and one commercial triterpenic acid, ursolic acid, were also chosen as mimetics of native antigens. Immunoenzymatic assays (ELISA) were performed for each compound to evaluate their potential as mimetics of native antigens of multiple sclerosis and Rett syndrome. The IgM levels in sera of patients affected by multiple sclerosis and Rett syndrome were measured and compared to normal blood donors. Concerning multiple sclerosis, no significant results were obtained for saponins, but in the case of Rett syndrome, interesting and surprising results were obtained. Indeed, the first hypothesis was that the glycosyl part of the molecule could be relevant for antibody recognition. In the case of Rett syndrome ursolic acid, an aglycone without any glycosidic part, demonstrated a good efficiency in IgM recognition. On the other hand, one triterpenic glycoside showed similar results. These results were discussed to define possible structure/activity relationships.