Introdução: A tuberculose (TB) é uma doença infectocontagiosa causada pelo Mycobacterium tuberculosis (MTb) que apresenta cerca de 80% predileção pelo acometimento pulmonar. Amostras de escarro são necessárias para identificar este microrganismo e o escarro induzido (EI) tem sido um método alternativo de obtenção destas amostras, porém tem apresentado, frequentemente, resultados negativos. A cultura, considerada padrão áureo, é mais morosa em seus resultados, portanto menos útil para guiar o diagnóstico. A reação em cadeia da polimerase (PCR) é a metodologia mais comum para o diagnóstico rápido da TB e poucos estudos avaliaram seu papel nas amostras de EI. Objetivo: Determinar acurácia diagnóstica do PCR para microbactéria no EI de pacientes com suspeita de TB pulmonar e descrever características sócio demográficas, dados clínicos, radiológicos, comorbidades e hábitos comportamentais. Métodos: Estudo prospectivo. Pacientes internados e ambulatoriais maiores de 18 anos com sintomas respiratórios sugestivos de tuberculose pulmonar (PTB) foram convidados a participar. Os sujeitos foram entrevistados utilizando-se um questionário padronizado e o EI foi coletado. Foram obtidas três amostras para baciloscopia direta e cultura. Obteve-se uma quarta amostra para o teste de PCR. Resultados: Foram avaliadas 116 amostras de escarro induzido. A sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo da PCR foram de 95,2%, 48,4%, 29,0% e 97,9%, respectivamente. A área sob a curva ROC foi de 0,72 para o teste de PCR (P <0,0001). Conclusões: Embora a especificidade da PCR possa ser subestimada, se considerarmos a PCR mais sensível do que os métodos de cultura utilizados pensaram que esses testes positivos para PCR significam falsos positivos. Os resultados de PCR devem ser sempre interpretados cuidadosamente em conjunto com informações clínicas. / Introduction: Induced sputum (IS) is an alternative method of obtaining sputum, but IS smears are frequently negative. Culture is more time-consuming in its results, and less useful to guide the diagnosis. Polymerase chain reaction (PCR) is the most common methodology for rapid diagnosis of tuberculosis and few studies evaluated its role in IS samples. Objective: The objective of this study is to determine the diagnostic yield of PCR for TB compared with culture in IS samples. Methods: Prospective study. Inpatients and outpatients > 18 years with respiratory symptoms suggestive of PTB were invited to participate. Subjects were interviewed using a standardized questionnaire, and collected IS. Three samples were obtained for AFB smear and culture. A fourth sample was obtained for PCR test. Results: A total of 116 induced sputum samples were evaluated. The sensitivity, specificity, positive predictive value, and negative predictive value of PCR were 95.2%, 48.4%, 29.0%, and 97.9%, respectively. The area under the ROC curve was 0.72 for the PCR test (P<0.0001). Conclusions: Although the PCR specificity could be underestimated, if we consider PCR to be more sensitive than the culture method used, we thought that these PCR positive tests means false-positives. PCR results should always be interpreted carefully in conjunction with clinical information.

L’hypertriglycéridémie (HTG) correspond à une accumulation des lipoprotéines riches en triglycérides (LRTG) dans la circulation plasmatique, conséquence d’une augmentation de leur synthèse ou plus classiquement décrit, d’une diminution de leur catabolisme. Dans près de 50% des cas, aucune cause génétique n’est identifiée chez les patients présentant une présentant une HTG sévère, aussi bien dans le cadre du syndrome de chylomicronémie familiale (FCS) que dans celui du syndrome de chylomicronémie multifactorielle (MCS). Pour améliorer nos connaissances et la caractérisation de ces patients, la conduction de corrélations phénotypes-génotypes précises grâce à une collaboration clinico-biologique étroite, ainsi que le développement d’outils de diagnostic moléculaire performants, demeurent un enjeu majeur. Premièrement, l’évaluation de la concentration pré-héparinique en LPL et l’activité post-héparinique 60 minutes après l’injection d’héparine chez 62 patients MCS caractérises génétiquement a permis la mise en évidence deux sous-groupes chez ces patients. Deuxièmement, le développement d’une stratégie séquençage de nouvelle génération permettant d’explorer simultanément les 9 gènes les plus prévalents dans les hypercholestérolémies, les hypocholestérolémies et les hypertriglycéridémies, a permis de détecter les variants nucléotidiques avec une sensibilité équivalente au séquençage Sanger mais aussi de détecter des grands réarrangements. L’ensemble des résultats souligne la complexité des mécanismes de régulation du métabolisme des LRTG et l’intérêt de l’étude des interactions gène-gène. Ainsi, ces travaux ont permis de mettre en évidence de nouvelles hypothèses à explorer pour la compréhension des mécanismes physiopathologiques des HTG sévères et d’améliorer les outils disponibles pour les études de corrélation génotype-phénotype / Hypertriglyceridemia (HTG) correspond to an increase of triglyceride-rich lipoproteins (TGRL) circulating concentration, as a consequence of an increase in the synthesis of or a decrease in their catabolism, most classically described. In nearly 50% of patients with severe hypertriglyceridemia (HTG), no genetic cause is identified, either in familial chylomicronemia syndrome (FCS) or in multifactorial chylomicronemia syndrome (MCS). To gain new insights and to improve patient’s characterization, it remains important to conduct accurate phenotype-genotype association studies through close collaboration with referent lipidologists, and to develop high-performance tools for molecular diagnosis. Firstly, the assessment of pre-heparin LPL concentration as well as LPL activity 60 minutes after heparin injection, enabled the identification of two subgroups within 62 genotyped MCS patients Secondly, the development of a new sequencing generation workflow exploring simultaneously the 9 most prevalent genes in dyslipidemia, allowed the detection of single nucleotide variations with sensitivity equivalent to Sanger sequencing, but also allowed the detection of copy number variations. Collective consideration of the results underlines the complexity of the regulation mechanisms of TGRL metabolism and the interest of gene-gene interactions study. Thus, the studies presented herein bring new hypothesis to explore for understanding the pathophysiological mechanisms of severe HTG and to improve molecular diagnosis tools available for phenotype-genotype association studies

Les entérovirus humains (EV) représentent la première cause des méningites aseptiques chez l'enfant. Le diagnostic de certitude repose sur la détection génomique de l'entérovirus par RT-PCR dans des échantillons de liquide céphalorachidien (LCR) est recommandée pour le diagnostic. La fièvre sans point d'appel et les maladies de type "sepsis" sont également des affections fréquentes chez les nourrissons (0 à 2 ans), qui peuvent être la conséquence d'une infection virale, en particulier à EV. Actuellement, le diagnostic des EV dans le sang est rarement établi dans la pratique courante.Les données concernant 1) l’existence d’une réplication de l’EV dans les leucocytes sanguins, 2) le niveau de charge virale de l’EV dans le sang et les échantillons de LCR et sa corrélation éventuelle avec l’intensité du processus inflammatoire réactionnel sont fragmentaires. Dans la première partie de cette thèse, l'objectif était d'évaluer la détection des EV par PCR dans des échantillons de sang de nouveau-nés, de nourrissons et d'enfants hospitalisés pour une fièvre isolée, un sepsis ou un syndrome méningé. Nous avons mené une étude observationnelle prospective multicentrique nationale (étude BLEDI) dans 35 départements de pédiatrie au cours de la période estivo-automnale (augmentation des circulations d'EV) en 2015-2016. Nos résultats ont montré que le taux de détection des EV dans le sang était significativement plus élevé que dans le LCR chez les nouveaux-nés et les nourrissons hospitalisés pour une fièvre isolée, un sepsis ou un syndrome méningé.Ces données ouvrent des perspectives pour un nouvel algorithme de diagnostic des fièvres inexpliquées chez les enfants âgés de moins de 2 ans. Dans le second volet de cette thèse, nous avons étudié de manière prospective la charge virale d'EV dans le sang et dans des échantillons de LCR de patients infectés. Nos résultats ont montré que la charge virale d'EV dans le sang variait selon le groupe d'âge, la présentation clinique et le type d'EV. Trois profils de cinétique d'infection comparant la charge virale dans le sang et le LCR ont été envisagés et sont en cours d'étude. Enfin, le dernier axe était de déterminer s'il existait une réplication de l’EV dans les leucocytes sanguins. Nous avons montré à partir d'échantillons de sang infectés in vitro par EV que la réplication de ces virus variait selon les génotypes d'EV. / Human enteroviruses (EV) are the most frequent cause of paediatric aseptic meningitis. Detection of enterovirus by PCR in cerebrospinal fluid (CSF) specimens is recommended for diagnosis. Fever without source and sepsis-like diseases are frequent affections in infants (0 to 2 years) which can be the consequence of a viral infection in particular to EV. At present, the daily routin diagnosis of EV in the blood is rarely performed. The concerning data 1) existence of EV replication in the blood leukocytes, 2) the EV viral load level in blood and CSF specimens and its possible relation with the intensity of the associated inflammatory process are fragmented. In the first part of this PhD thesis, the aim was to assess detection of enterovirus by PCR in blood specimens of newbrons, infants, and children with fever without source, sepsis-like disease or suspected meningitis. We did a prospective, multicentre, observational study (BLEDI study) at 35 paediatric departements during the seasonal period of increased EV circulation in 2015-2016. Our results showed that detection of EV was significantly higher in the blood than in the CSF from newborns and infants admitted with fever without source or sepsis-like disease. These data open up the perspectives for a new diagnostic algorithm of febrile illness in patients aged 2 years or younger. We also explored prospectively EV viral load in blood and in CSF specimens of infected patients. Our results showed that EV viral load in the blood varied by age group, clinical presentation and EV type. Three profiles of infection kinetics comparing EV viral load in the blood and the CSF have been considered and are under study. Finally, we explored the hypothesis of an EV replication in the blood leukocytes. We showed from blood samples infected in vitro by EV that the replication of these viruses varied by EV genotypes.

O objetivo principal deste estudo foi avaliar a frequência de cepas do HBV com mutações de resistência aos análogos nucleos(t)ídeos (AN) utilizados no tratamento da hepatite B entre indivíduos cronicamente infectados, não submetidos a tratamento, procedentes de diferentes regiões do Brasil. Além disso, foram avaliadas a presença de mutações que alteram a antigenicidade do HBsAg promovendo escape dos anticorpos anti-HBs; mutações nos genes pré-core/core e a associação dos diferentes subgenótipos com as mutações encontradas e características demográficas e laboratoriais dos pacientes. Foram incluídas 779 amostras de soro de pacientes com infecção crônica pelo HBV e virgens de tratamento com AN ou interferon, as quais foram coletadas no período de 2006 a 2011. Os pacientes eram procedentes dos seguintes estados brasileiros: Pará, Maranhão, Bahia, Minas Gerais, São Paulo, Paraná e Rio Grande do Sul. O DNA do HBV foi extraído das amostras de soro utilizando o Kit QIAamp DNA Blood Mini Kit (Qiagen) e posteriormente foi realizada a amplificação das regiões S/polimerase (S/P) e pré-core/core (PCC) do genoma viral por nested PCR. O fragmento amplificado foi submetido a sequenciamento direto em sequenciador automático de DNA (ABI 3500) e as sequências obtidas foram analisadas para identificação dos genótipos e subgenótipos do HBV, pesquisa de mutações na polimerase, no HBsAg e nos genes pré-core/core. A região S/Pol foi amplificada e sequenciada com sucesso em 702 amostras, as quais foram incluídas para atender aos objetivos deste estudo. Entre as 702 amostras analisadas sete genótipos e 12 subgenótipos do HBV foram identificados. O subgenótipo A1 foi o mais frequente (63,7%, 447/702), seguido pelo HBV/D3 (14,5%, 102/702). Os demais genótipos e subgenótipos encontrados e suas frequências foram as seguintes: A2 (3,3%, 23/702), A3 (0,1%, 1/702), B1 (0,1%, 1/702), B2 (0,1%, 1/702), C2 (0,9%, 6/702), D1 (0,9%, 6/702), D2 (4,6%, 32/702), D4 (5,1%, 36/702), D com subgenótipo não identificado (0,7%, 5/702), E (0,6%, 4/702), F2a (4,6%, 32/702), F4 (0,4%, 3/702), e G (0,4%, 3/702). Cepas do HBV com mutações de resistência (rtS202G, rtM204V/I, rtA194T, rtM250I, rtA181T/S, rtT184S) associadas ou não a mutações compensatórias (rtL80I, rtV173L, rtL180M, rtV207I) foram identificadas em 1,6% (11/702) das amostras analisadas. Cepas com mutações potencialmente associadas com resistência ao adefovir (rtS85A, rtL217R, rtI233V, rtN238T, rtN238D, rtN248H, rtV214A,e rtQ215S) ou ao entecavir (rtS219A) foram identificadas em 7,7% (54/702) e 2,6% (16/702) dos pacientes, respectivamente. Cinquenta e sete (8,5%) amostras apresentaram cepas do HBV com mutações na principal região hidrofílica do HBsAg previamente relacionadas com escape dos anticorpos anti-HBs ou com prejuízo na secreção do HBsAg. Foram feitas análises estatísticas para avaliar a correlação entre os subgenótipos do HBV mais frequentes na casuística (A1, A2, D1, D2, D3, D4 e F2a) e a presença de mutações nos genes PCC. Dentre as mutações nos genes PCC associadas com redução ou falha na expressão do HBeAg, as mutações A1762T/T1764A estiveram associadas aos subgenótipos A1 e F2a; G1862T e mutações nas posições 1809-1812 ao subgenótipo A1; G1896A e/ou G1899A aos subgenótipos D2, D3 e D4. Mutações associadas com evolução da doença foram detectadas e entre essas as mutações C1766T e T1768A estiveram associadas aos subgenótipos A1 e F2a, e a mutação G1888A foi associada ao subgenótipo A1. As cepas do HBV que circulam nas diferentes regiões brasileiras estudadas apresentam grande variabilidade genética e a distribuição dos genótipos e subgenótipos reflete a formação histórica de cada região e do fluxo migratório mais recente. A frequência de cepas do HBV com mutações de resistência aos AN circulando entre pacientes virgens de tratamento com esses medicamentos nas diferentes regiões do Brasil estudadas é baixa, sendo que o perfil de mutações que confere resistência total à lamivudina e parcial ao entecavir parece ser o mais disseminado. Embora tenham sido detectados casos de infecção com cepas do HBV portando mutações com grande impacto na antigenicidade dessa proteína todas as amostras apresentaram HBsAg detectável. Pacientes com HBeAg negativo foram mais frequentes na casuística estudada, independente do subgenótipo. As mutações encontradas nos genes PCC sugerem que há perfis de mutações diferentes envolvidos na negatividade do HBeAg para cada subgenótipo / The main aim of this study was to evaluate the frequency of HBV strains harboring mutations that confer resistance to nucleos(t)ide analogues (NA) used to hepatitis B treatment among treatment-naïve patients with chronic hepatitis B from different Brazilian region. Furthermore, we evaluated the presence of mutations that alter the antigenicity of HBsAg causing anti-HBs escape; mutations in genes pre-core/core and the association of different subgenotypes with the mutations detected and demographic and laboratory characteristics of the patients. Serum samples from 779 treatment-naïve patients with chronic HBV infection were included in this study. The samples were collected between 2006 to 2011 and the patients were from the following states: Pará, Maranhão, Bahia, Minas Gerais, São Paulo, Paraná and Rio Grande do Sul. HBV DNA was extracted from serum samples using the QIAamp DNA Blood Mini Kit (Qiagen) and amplification of S/polymerase (S/Pol) and pre-core/core (PCC) regions were performed by nested PCR. The amplified PCR products were submitted to sequencing in an automatic DNA sequencer (ABI 3500). The sequences obtained were analyzed to classify HBV genotypes/subgenotypes and to analyze the presence of mutations. S/Pol region was amplified and sequenced successfully from 702 samples, which were included in this study. Among these 702 samples, seven genotypes and 12 subgenotypes have been identified. HBV subgenotype A1 was the most frequent (63.7%, 447/702), followed by HBV/D3 (14.5%; 102/ 702). The remaining genotypes and subgenotypes identified and their frequencies were as follows: A2 (3.3%, 23/702), A3 (0.1%, 1/702), B1 (0.1%, 1/702), B2 (0.1%, 1/702), C2 (0.9%, 6/702), D1 (0.9%, 6/702), D2 (4.6%, 32/702), D4 (5.1%, 36/702), D unclassified subgenotype (0.7%, 5/702), E (0.6%, 4/702), F2a (4.6%, 32/702), F4 (0.4%, 3/702), and G (0.4%, 3/702). HBV strains harboring mutations conferring NA resistance alone (rtS202G, rtM204V/I, rtA194T, rtM250I, rtA181T/S, rtT184S) or combined with compensatory mutations (rtL80I, rtV173L, rtL180M, rtV207I) were identified in 1.6% (11/702) of the patients. Isolates harboring mutations potentially associated with adefovir resistance (rtS85A, rtL217R, rtI233V, rtN238T, rtN238D, rtN248H, rtV214A, and rtQ215S) or entecavir resistance (rtS219A) were identified in 7.7% (54/702) and 2.6% (16/702) of the patients, respectively. HBV with HBsAg mutations previous related with anti-HBs escape or impaired secretion were detected in 8.5% (57/702) of the samples. Statistical analyzes were performed to assess the correlation between the more frequent HBV subgenotypes found in this study (A1, A2, D1, D2, D3, D4 and F2a ) and mutations in PCC genes. Among the mutations found in these genes that were associated with reduction or failure in HBeAg synthesis, A1762T/T1764A mutations were associated to subgenotypes A1 and F2a; G1862T and mutations at positions 1809-1812 to subgenotype A1; G1896A and/or G1899A to subgenotypes D2, D3 and D4. Other mutations associated with disease progression were found: C1766T and T1768A mutations were associated with subgenotypes A1 and F2a, and the G1888A mutation was associated with subgenotype A1. HBV strains circulating in different Brazilian regions studied showed high genetic variability and distribution of genotypes and subgenotypes reflects the population formation history of each region and the occurrence of recent events of migration. The frequency of HBV strains with NA resistance mutations circulating among treatment-naive patients in different regions of Brazil studied is low and the profile of mutations that confer total resistance to lamivudine and partial resistance to entecavir is more widespread. Although some cases of infection have been detected with HBV strains carrying mutations associated with major impact on the antigenicity of this protein, all samples had detectable HBsAg. HBeAg negative cases were more frequent in the studied population, regardless of subgenotype. Different pattern of mutations were found in PCC genes, suggesting that different mechanisms are involved in HBeAg negativity for each subgenotype

O vírus da cinomose canina (VCC) é um patógeno viral, altamente, contagioso que pode causar doença sistémica letal, em cães e outros carnívoros em toda parte do mundo. Os cães afetados podem apresentar sintomas gastrentéricos, respitatórios e nervosos. As manifestações clínicas da doença inclue depressão, diarréia, vómito, desidratação, hiperqueratose dos coxins e focinho e espasmos musculares ou paresia de membros pélvicos, a qual pode persistir por longos períodos. Cães infectados, com sintomas clínicos de VCC, foram estudados para detecção do RNA viral pela técnica de PCR e Nested-PCR. Neste estudo, amplificou-se o gene da nucleoproteína (NP) em células mononucleares do sangue periférico (linfócitos), urina e saliva, de cães infectados com VCC, para detectar o genoma do mesmo, por RT-PCR, em diferentes amostras clínicas. A identificação do RNA viral foi concluída com sucesso, pelo método de RT-PCR, utilizando 2 pares de \"primers\" específicos do gene da nucleoproteína (NP). A técnica de RT-PCR, descrita neste etudo, pode ser um sistema de ensaio útil para determinar se cães suspeitos de infecção, por VCC, tenha níveis detectáveis de genes. Os resultados demonstram que a técnica de RT-PCR é exequível para o diagnóstico laboratorial de cinomose canina. / Canine distemper vírus (CDV) is a highly contagious Viral pathogen which may cause lethal systemic in dogs and other carnivores throughout the world. Affected dogs show gastrointestinal and respiratory clinical slgns, and frequently develop clinical signs in the central nervous system (CNS). Clinical manifestations of the disease include depression, progressive loss of weight, dehydration, hyperkeratosis of the foot pads and nose, nervous symptoms and muscular spasms or posterior paralysis which may perslst for long periods. Infected dogs with clinical symptoms for CDV, were by detection of viral RNA by Polymerase Chaln Reaction (PCR) and Nested PCR. In this study,w e determinebdy the RT-PCRth e presenceo f nucleoprotein (NP) gene in peripheral blood mononuclear cells, urine and saliva from dogs infected with CDV. The goals of this study was to detect CDV renome by RT-PCR in different clinical samples. In this study, Identificatlon of NP mRNA was successfully achieved by using the RT-PCR method with two sets of NP gene specific primers. The RT-PCR technique described in thls study, may provide a useful assay system to determine whether the dogs suspected of CDV infection have detectable leveis of CDV genes. The results demonstrate that RT-PCR technique is rapid, sensitivity and specificity for vírus diagnosis.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-08-05T14:19:01Z No. of bitstreams: 1 2010 - Raquel Saucier Gomes.pdf: 7033294 bytes, checksum: d1538edd22d0ad5fcbf8c9c1e86f330e (MD5) / Made available in DSpace on 2016-08-05T14:19:01Z (GMT). No. of bitstreams: 1 2010 - Raquel Saucier Gomes.pdf: 7033294 bytes, checksum: d1538edd22d0ad5fcbf8c9c1e86f330e (MD5) Previous issue date: 2010-02-09 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico, CNPq. / This study aimed to diagnose and characterize genetically the species and genotypes of Cryptosporidium in stool samples from poultry and exotic birds marketed in Rio de Janeiro involving possible risk factors for infection. We analyzed 180 poultry sold in local markets and 103 exotic birds from breeding and pet shops. For analysis, the DNA extracted from fecal sample suspension was used to amplify the 18S rDNA sequences by nested-PCR technique. The amplicons generated were subjected to RFLP using the enzymes SspI and VspI, and sequencing and phylogenetic analysis, to confirm the species. Different species of Cryptosporidium were indentified in faecal samples of poultry and exotic birds. In the analysis of sites of enzymatic cutting products of nested-PCR gene 18SR DNA, the species C. bailey was the only one that displayed a characteristic cut-off, but other samples were. The following species were diagnosed: C. baileyi (GU082387) infecting ducks (Anas platyrhynchus domesticus), C. parvum in quails (Coturnix japonica) (GU082384 and GU082386), chicks (Gallus gallus domesticus) (GU082390 and GU082391), duck (GU082388) and Manon (Lonchura striata domestica) (GU074390). The Avian genotype III was identified in caulker (Lonchura padda oryzivora) (GUO74384) and cockatiel (Nymphicus hollandicus) (GU074385, GU074386 and GU074387). The sequences of canaries (Serinus canarius) received access numbers GU074388 and GU074389, but it was not possible to identify the species of Cryptosporidium, because of the large genetic distance between them and those already deposited in GenBank, suggesting a new genotype or a new specie. Although C. baileyi and Avian genotype III are common in birds, the diagnosis of C. parvum is a worrying finding, since this species are more associated with mammals. Birds can be considered as reservoirs and disseminators of environmental infective form of the parasite, allowing the infection to a large number of species of hosts, including in the man in the epidemiological chain. / O presente trabalho teve por objetivo diagnosticar e caracterizar geneticamente esp?cies e/ou gen?tipos de Cryptosporidium em amostras fecais de aves dom?sticas e ex?ticas comercializadas no Estado do Rio de Janeiro, associando poss?veis fatores de risco da infec??o. Foram analisadas 180 aves dom?sticas comercializadas em mercados locais e 103 aves ex?ticas de criadouros e petshops. Para as an?lises, o DNA extra?do de suspens?o de amostra fecal foi utilizado na amplifica??o das seq??ncias do 18S rDNA atrav?s da t?cnica Nested-PCR. Os amplicons gerados foram submetidos ? RFLP, utilizando as enzimas SspI e VspI, e ao seq??nciamento, para a confirma??o das esp?cies. Foram identificadas esp?cies de Cryptosporidium em amostras fecais de aves dom?sticas e ex?ticas. Durante as an?lises dos s?tios de corte enzim?tico dos produtos da Nested-PCR do gene 18Sr DNA, a esp?cie C. baileyi foi a ?nica que apresentou padr?o de corte caracter?stico, por?m nas demais amostras foi necess?ria a confirma??o atrav?s do sequenciamento e estudo filogen?tico. Foi diagnosticado C. baileyi (GU082387) infectando patos (Anas platyrhynchus domesticus); C. parvum em codornas (Coturnix coturnix japonica) (GU082384 e GU082386), pintos (Gallus gallus domesticus) (GU082390 e GU082391), pato (GU082388) e manon (Lonchura striata domestica) (GU074390). O gen?tipo avi?rio III foi identificado pela primeira vez em calafate (Lonchura padda oryzivora) (GUO74384) e em calopsita (Nymphicus hollandicus) (GU074385, GU074386 e GU074387). As seq??ncias de can?rios (Serinus canarius) receberam os n?meros de acesso GU074388 e GU074389, por?m n?o foi poss?vel a identifica??o da esp?cie de Cryptosporidium, devido ? grande dist?ncia gen?tica entre elas e aquelas j? depositadas no GenBank, sugerindo novo gen?tipo ou uma nova esp?cie . Embora C. baileyi e o gen?tipo Avi?rio III sejam comuns em aves, o diagn?stico de C. parvum ? um achado preocupante, j? que esta esp?cie est? mais associada com mam?feros. Aves podem ser consideradas como reservat?rios e disseminadoras ambientais da forma infectante do protozo?rio, possibilitando a infec??o para um amplo n?mero de esp?cie de hospedeiros incluindo, nesta cadeia epidemiol?gica, o ser humano.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-05-03T21:15:13Z No. of bitstreams: 1 2016 - Daniel Guimar?es Ubiali.pdf: 7253233 bytes, checksum: ce10a8e3d4ef67ad93a0034844382ed3 (MD5) / Made available in DSpace on 2017-05-03T21:15:13Z (GMT). No. of bitstreams: 1 2016 - Daniel Guimar?es Ubiali.pdf: 7253233 bytes, checksum: ce10a8e3d4ef67ad93a0034844382ed3 (MD5) Previous issue date: 2016-10-26 / CAPES / For investigation of Mycobacterium avium subsp. paratuberculosis infection 34 buffaloes properties or ranches in Northeastern Brazil were visited for paratuberculosis diagnosis. Investigations included herd evaluations, inspection of facilities and pastures, obtainment of flock history, clinical examination of suspicious animals and collecting samples for diagnosis. Samples were obtained from 26 farms or ranches, including two slaughterhouses and a quarantine area in six states. Approximately 15,600 buffalos, including males and females of the Murrah, Mediterranean and Jafarabadi breeds as well as their crossbreeds, were evaluated for meat, dairy and mixed properties with semi-intensive or extensive regimes. For diagnostic purposes, necropsies, histopathological and immunohistochemical exams and Ziehl-Neelsen tests of fecal smears and scraped intestinal mucosa were performed. Polymerase chain reaction (PCR) methods were applied to samples of feces, milk, mesenteric nodes and intestines. This exams allowed us to identify eigth new Johne?s disease outbreaks, which, together with those previously identified by our staff, allow us to infer that the disease is being dispersed in the Brazilian Northeast, similar to what is occurring with bovine herds in other areas of the country. The increase in the number of positive farms is a consequence of the ignorance of farmers, inadequate health management, free trade of ruminants and lack of a official control program in the country. This study alerts for risk of commercialization of dairy products for human consumption, reinforces the importance of research on paratuberculosis in Brazil and contributes to the understanding of the factors that work together to increase the number of paratuberculosis cases in the Brazilian Northeast. / Com o objetivo de identificar focos e estudar a epidemiologia e o diagn?stico da paratuberculose em b?falos na regi?o Nordeste do Brasil foram realizadas visitas e ou examinados material provenientes de 34 propriedades com suspeita cl?nica da doen?a. Obtivemos material biol?gico de sete estados da regi?o Nordeste do Brasil (Maranh?o, Cear?, Para?ba, Pernambuco, Alagoas e Bahia). Dos rebanhos foi obtido o hist?rico, realizou-se avalia??o cl?nica e inspe??o das instala??es e pastagens. Para a confirma??o do diagn?stico foi coletado material para exames laboratoriais em 26 propriedades ou cria??es de b?falos, entre estes, dois matadouros e um quarenten?rio. Foram rebanhos cujo somat?rios de b?falos era de aproximadamente 15.600 b?falos, das ra?as Murrah, Mediterr?neo, Jafarabadi e seus mesti?os, com aptid?o para corte, leite ou mista, em propriedades com regimes semi-intensivo, extensivo ou extrativista. Foram realizadas 22 necropsias e coleta de material para exames histopatol?gicos e imuno-histoqu?micos, al?m de colora??o de Ziehl-Neelsen em esfrega?os de fezes, raspados de mucosa intestinal e fragmentos de linfonodo mesent?rico e de intestino que apresentavam les?es sugestivas da doen?a. Para a realiza??o da rea??o em cadeia da polimerase (PCR) foram utilizadas amostras de fezes, leite, linfonodos mesent?ricos e intestinos. Estes exames permitiram identificar oito focos de paratuberculose, o que nos permite inferir que a doen?a est? se dispersando na regi?o Nordeste do Brasil a exemplo do que est? acontecendo em outras regi?es do pa?s com o rebanho bovino. O desconhecimento da doen?a, o manejo inadequado, o com?rcio n?o regulamentado de b?falos e a falta de um programa de controle voltado para a realidade da regi?o facilitam a dispers?o do agente e s?o fatores que contribuem para o aumento do n?mero de focos no pa?s. Os resultados desta pesquisa contribuem com a epidemiologia e o diagn?stico da doen?a em b?falos e auxilia na compreens?o dos fatores que colaboram para a ocorr?ncia crescente do n?mero de casos desta doen?a.

O vírus da cinomose canina (VCC) é um patógeno viral, altamente, contagioso que pode causar doença sistémica letal, em cães e outros carnívoros em toda parte do mundo. Os cães afetados podem apresentar sintomas gastrentéricos, respitatórios e nervosos. As manifestações clínicas da doença inclue depressão, diarréia, vómito, desidratação, hiperqueratose dos coxins e focinho e espasmos musculares ou paresia de membros pélvicos, a qual pode persistir por longos períodos. Cães infectados, com sintomas clínicos de VCC, foram estudados para detecção do RNA viral pela técnica de PCR e Nested-PCR. Neste estudo, amplificou-se o gene da nucleoproteína (NP) em células mononucleares do sangue periférico (linfócitos), urina e saliva, de cães infectados com VCC, para detectar o genoma do mesmo, por RT-PCR, em diferentes amostras clínicas. A identificação do RNA viral foi concluída com sucesso, pelo método de RT-PCR, utilizando 2 pares de \"primers\" específicos do gene da nucleoproteína (NP). A técnica de RT-PCR, descrita neste etudo, pode ser um sistema de ensaio útil para determinar se cães suspeitos de infecção, por VCC, tenha níveis detectáveis de genes. Os resultados demonstram que a técnica de RT-PCR é exequível para o diagnóstico laboratorial de cinomose canina. / Canine distemper vírus (CDV) is a highly contagious Viral pathogen which may cause lethal systemic in dogs and other carnivores throughout the world. Affected dogs show gastrointestinal and respiratory clinical slgns, and frequently develop clinical signs in the central nervous system (CNS). Clinical manifestations of the disease include depression, progressive loss of weight, dehydration, hyperkeratosis of the foot pads and nose, nervous symptoms and muscular spasms or posterior paralysis which may perslst for long periods. Infected dogs with clinical symptoms for CDV, were by detection of viral RNA by Polymerase Chaln Reaction (PCR) and Nested PCR. In this study,w e determinebdy the RT-PCRth e presenceo f nucleoprotein (NP) gene in peripheral blood mononuclear cells, urine and saliva from dogs infected with CDV. The goals of this study was to detect CDV renome by RT-PCR in different clinical samples. In this study, Identificatlon of NP mRNA was successfully achieved by using the RT-PCR method with two sets of NP gene specific primers. The RT-PCR technique described in thls study, may provide a useful assay system to determine whether the dogs suspected of CDV infection have detectable leveis of CDV genes. The results demonstrate that RT-PCR technique is rapid, sensitivity and specificity for vírus diagnosis.

Les techniques de biologie moléculaire ont pris au cours des 20 dernières années une place importante dans le diagnostic direct des pathogènes viraux. Notre travail a porté sur la mise en place et le développement d’une plate-forme de biologie moléculaire, au sein du laboratoire de virologie de l’hôpital de la Timone, pour répondre aux demandes et contraintes du diagnostic en milieu hospitalier. L’organisation de cette plate-forme a nécessité plusieurs étapes : la prévention des risques de contamination, l’aliquotage et le stockage des réactifs, l’automatisation des techniques d’extraction des acides nucléiques, la mise au point de témoins positifs synthétiques et de témoins internes et l’optimisation des protocoles de PCR. Cette approche optimisée du diagnostic moléculaire des infections virales a été appliqué notamment à la détection de la grippe pandémique A/H1N1v dans les laboratoires de routine hospitalière et d’urgence « Point Of Care ». La mise en place de cette plate-forme a fait progresser de manière considérable le diagnostic moléculaire du laboratoire. Elle nous permet actuellement de détecter un grand nombre de pathogènes (>80) et de réaliser des tests dans un format à haut débit (≈40 000 tests/an). Au total, cette plateforme est au coeur de la capacité du laboratoire pour réagir de manière rapide aux évènements d'émergence en mettant en place rapidement des procédures diagnostiques standardisées. Ces techniques ont été transférées à de nombreux autres laboratoires de virologie partenaires nationaux et internationaux. Nous envisageons maintenant son utilisation dans une approche syndromique avec notamment, le développement du diagnostic des virus respiratoires. / Molecular biology techniques have taken an important role in the direct diagnosis of viral pathogens over the last 20 years. Our work focused on establishing and developing a platform for molecular diagnosis in the laboratory of Virology (Timone Hospital) to meet the demands and constraints of diagnosis in hospitals. The organization of this platform required several steps: prevention of contamination risks, aliquoting and storage of reagents, automation techniques of nucleic acid extraction, development of synthetic positive controls and internal controls and optimization of PCR protocols. This optimized approach of the molecular diagnosis of viral infections has particularly been applied to the detection of pandemic influenza A/H1N1v in hospital laboratories for routine and emergency "Point Of Care." The implementation of this platform has significantly improved molecular diagnosis in our laboratory. It currently allows us to detect a large number of pathogens (> 80) and perform tests in a high-throughput (≈ 40,000 tests per year). In total, this platform is at the heart of the laboratory capacity to react quickly to emerging events by rapidly implementing standardized procedures. These techniques have been transferred to many other partners’ laboratories nationally and internationally. We are now considering its use in a syndromic approach including the development of the diagnosis of respiratory viruses.

Magnetic resonance imaging (MRI) is regularly used to obtain anatomical images, greatly advancing biomedical research and clinical health care today, but its full potential in providing functional, physiological, and molecular information is only beginning to emerge. The goal of magnetic resonance molecular imaging is to utilize MRI to acquire information on the molecular level. This dissertation is focused on ways to increase the use of MRI for molecular imaging using superparamagnetic iron oxide (SPIO) nanoparticle induced MRI contrast. This work is divided into three main sections: <B>1)<I> Elucidation of the contribution of size and coating properties to magnetic nanoparticle induced proton relaxation.</I></B> To maximize contrast generated without increasing particle size, new methods to increase effects on relaxivity must be developed. Experimental data obtained on a new class of biocompatible particles are presented, along with simulated data. The effects of coating size, proton exchange, and altered diffusion are examined. Simulations are presented confirming the effect of particle coatings on clustering-induced relaxivity changes, and an experimental system demonstrating the clustering effect is presented. <B>2)<I> Development of a diffusion-dependent, off-resonance imaging protocol for magnetic nanoparticles.</I></B> This work demonstrates an alternative approach, off-resonance saturation (ORS), for generating contrast sensitive to SPIO nanoparticles. This method leads to a calculated contrast that increases with SPIO concentration. Experimental data and a mathematical model demonstrate and characterize this diffusion-dependent, off-resonance effect. Dependence on off-resonance frequency and power are also investigated. <B>3)<I> Development of a genetic MRI marker via in vivo magnetic nanoparticle synthesis.</I></B> This work seeks to provide a gene expression marker for MRI based on bacterial magnetosomes, tiny magnets produced by naturally occurring magnetotactic bacteria. Here, <I>magA</I> is expressed in a commonly used human cell line, 293FT, resulting in the production of magnetic, iron oxide nanoparticles by these cells. MRI shows these particles can be formed <I>in vivo</I> utilizing endogenous iron and can be used to visualize cells positive for <I>magA</I>. These results demonstrate <I>magA</I> alone is sufficient to produce magnetic nanoparticles and that it is an appropriate candidate for an MRI reporter gene.